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  • 99
    New England Biolabs mnase
    AGO2 knock-down affects nucleosome occupancy at TSSs bound by SWI/SNF. ( a ) HeLaS3 cells were transfected with a control siRNA (siCTRL) or a pool of AGO2 siRNA (siAGO2). Down-regulation of AGO2 protein was verified by western blot. GAPDH was used as loading control. ( b ) Chromatin from siCTRL- or siAGO2-treated HeLaS3 cells was digested by <t>MNase</t> and recovered <t>DNA</t> fragments were sequenced. Nucleosome occupancy profile for siCTRL and siAGO2 cells was plotted for TSSs with at least 30 swiRNAs (siCTRL, black line; siAGO2, green line). The occupancy at the nucleosome +1 (arrow) is reduced in AGO2 knock-down cells. ( c ) Bars height represents percent reduction of nucleosome occupancy (siAGO2 versus siCTRL) at TSS ±150 nt overlapped by at least the indicated number of swiRNAs (green), IgG-IP ‘other sRNAs’ (black) and AGO1-associated ‘other sRNAs’ (purple). ** P value
    Mnase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 730 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher mnase reaction buffer
    AGO2 knock-down affects nucleosome occupancy at TSSs bound by SWI/SNF. ( a ) HeLaS3 cells were transfected with a control siRNA (siCTRL) or a pool of AGO2 siRNA (siAGO2). Down-regulation of AGO2 protein was verified by western blot. GAPDH was used as loading control. ( b ) Chromatin from siCTRL- or siAGO2-treated HeLaS3 cells was digested by <t>MNase</t> and recovered <t>DNA</t> fragments were sequenced. Nucleosome occupancy profile for siCTRL and siAGO2 cells was plotted for TSSs with at least 30 swiRNAs (siCTRL, black line; siAGO2, green line). The occupancy at the nucleosome +1 (arrow) is reduced in AGO2 knock-down cells. ( c ) Bars height represents percent reduction of nucleosome occupancy (siAGO2 versus siCTRL) at TSS ±150 nt overlapped by at least the indicated number of swiRNAs (green), IgG-IP ‘other sRNAs’ (black) and AGO1-associated ‘other sRNAs’ (purple). ** P value
    Mnase Reaction Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    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: 86/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    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: 99/100, based on 860 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    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: 97/100, based on 417 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    BioShop micrococcal nuclease s7
    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
    Micrococcal Nuclease S7, supplied by BioShop, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    US Biological Life Sciences p1 microccocal nuclease
    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
    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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    94
    Roche mnase buffer
    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 Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc 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 Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    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: 93/100, based on 255 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    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: 84/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Millipore 25 u microccocal nuclease
    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.
    25 U Microccocal Nuclease, supplied by Millipore, used in various techniques. Bioz Stars score: 87/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    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: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    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: 93/100, 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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    87
    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: 87/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    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: 86/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    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: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    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: 87/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    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: 94/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher mnase digestion 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.
    Mnase Digestion Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    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: 96/100, based on 584 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    GE Healthcare 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 GE Healthcare, used in various techniques. Bioz Stars score: 88/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Illumina Inc mnase
    Phased nucleosome arrays flanked TSSs of rice genes. (A) Nucleosome positioning profile associated with active genes. Phased nucleosome arrays are detectable after the TSSs. (B) Nucleosome positioning profile associated with non-expressed genes. Phased nucleosome arrays are detected on either side of the TSSs. (C) Distribution of DHS length for five different DHS categories. Note: the length of DHSs associated with proximal promoters (black line) are more variable than the lengths of other DHSs. (D) Heatmap of nucleosome positioning associated with active genes. Left panel: All expressed genes were sorted by the length of DHSs located in proximal promoters. The 5′ ends of the <t>MNase-seq</t> reads were mapped within 1 kb upstream and 1 kb downstream of the TSS of each gene to show the boundaries of <t>nucleosomes</t> core and linker. The red line on the left heatmap indicates the boundaries of DHSs. With the same order of the genes as in the left panel, the 5′ ends of DNase-seq reads (middle panel) and the fragments per kilobase of exon per million fragments mapped (FPKM) value log10 transformation (right panel) were mapped to show the DNase I sensitivity and the expression level of each gene, respectively.
    Mnase, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 75 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology mnase
    Phased nucleosome arrays flanked TSSs of rice genes. (A) Nucleosome positioning profile associated with active genes. Phased nucleosome arrays are detectable after the TSSs. (B) Nucleosome positioning profile associated with non-expressed genes. Phased nucleosome arrays are detected on either side of the TSSs. (C) Distribution of DHS length for five different DHS categories. Note: the length of DHSs associated with proximal promoters (black line) are more variable than the lengths of other DHSs. (D) Heatmap of nucleosome positioning associated with active genes. Left panel: All expressed genes were sorted by the length of DHSs located in proximal promoters. The 5′ ends of the <t>MNase-seq</t> reads were mapped within 1 kb upstream and 1 kb downstream of the TSS of each gene to show the boundaries of <t>nucleosomes</t> core and linker. The red line on the left heatmap indicates the boundaries of DHSs. With the same order of the genes as in the left panel, the 5′ ends of DNase-seq reads (middle panel) and the fragments per kilobase of exon per million fragments mapped (FPKM) value log10 transformation (right panel) were mapped to show the DNase I sensitivity and the expression level of each gene, respectively.
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    94
    ATUM mnase digested dna
    Nucleosome positioning at a (CAG) 85 repeat is not altered in the absence of H2A.1 or H2A.2. A ) Indirect end-labeling of nucleosomal <t>DNA</t> upstream of the CAG repeat. <t>MNase</t> (0, 0.25, 2.5, and 7.5 units) digested DNA was run in 1.5% agarose with ethidium bromide (left) and Southern blotted (right) using a probe ~100 bp proximal to the CAG repeat (red line Figure 1—figure supplement 1A ). Ovals represent nucleosome positions. The experiment was repeated six times; a representative blot is shown. ( B ) Illumina array mapping of nucleosome protection at the CAG repeat. Mononucleosomal DNA from strains containing the (CAG) 85 repeats was hybridized to a custom array of 30-mer probes spanning 425 bp upstream of the repeat to 436 bp downstream of the repeat in YAC CF1. Probes 14–16 contain CAG repeats; probe 15 is composed purely of CAG repeats (probe sequences in Supplementary file 3 ). Error bars represent standard deviation of 2–3 independent experiments.
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    93
    Roche micrococcal nuclease mnase
    Nucleosome reassembly at the TSS is the initiating event in MLH1 resilencing. A , Regions assayed for nucleosome occupancy using <t>MNase-qPCR</t> (Regions I–IX) and NOMe-Seq (Regions N1 and N2). Shown beneath the gene schematic is NOMe-Seq data from untreated RKO cells at Region N1. Black arrows indicate the MLH1 and EPM2AIP1 TSS. Bottom panel represents GpC accessibility. Black circles = GpC dinucleotides methylated/accessible to the GpC methyltransferase M. Cvi PI. White circles = GpC dinucleotides not methylated/inaccessible to GpC methyltransferase. Pink shading indicates regions of inaccessibility of ≥150 bp. Asterix = region of M. Cvi PI accessibility. B , Relative nucleosome levels in untreated RKO cells at the indicated regions (black bars labeled Regions I–IX) as determined by MNase-qPCR. Drawn to scale with schematic shown in A. Error bars = SD. C and D , qPCR results showing changes in relative nucleosome levels at Regions III and VI following decitabine exposure. E and F , MLH1 gene expression (E) and promoter bisulfite pyrosequencing (F), reproduced from Figure 2A and D for ease of comparison with nucleosome levels. G–I , NOMe-Seq analysis of the MLH1 promoter at Region N2 in SW620 (F) and RKO (G,H) cells at the indicated treatment points. Black filled triangles = methylated CpG dinucleotides; white filled triangles = unmethylated CpG dinucleotides. See also Figure S2 .
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    Millipore micrococcal nuclease mnase
    shRNA-mediated inactivation of ATRX does not alter subtelomeric chromatin accessibility to <t>MNase.</t> (A) Chromatin isolated from 8-MG-BA glioma cells in which ATRX had been inactivated (shATRX) or not (shscrambled [shSCR]) was digested with MNase for the indicated times. (Left gel) Ethidium bromide (EtBr) staining of bulk chromatin. (Right gel) Southern blot with subtelomeric probe. (Far right) Quantification of the data. The signals obtained for mononucleosomes were normalized to the total signals measured for each time point (EtBr or Southern blot). (B) Chromatin samples from shATRX or shSCR 8-MG-BA cells were digested for 5 min with the indicated amounts of MNase (milliunits per microgram of DNA). (Far right) Quantification of the data.
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    Millipore staphylococcus aureus mnase
    shRNA-mediated inactivation of ATRX does not alter subtelomeric chromatin accessibility to <t>MNase.</t> (A) Chromatin isolated from 8-MG-BA glioma cells in which ATRX had been inactivated (shATRX) or not (shscrambled [shSCR]) was digested with MNase for the indicated times. (Left gel) Ethidium bromide (EtBr) staining of bulk chromatin. (Right gel) Southern blot with subtelomeric probe. (Far right) Quantification of the data. The signals obtained for mononucleosomes were normalized to the total signals measured for each time point (EtBr or Southern blot). (B) Chromatin samples from shATRX or shSCR 8-MG-BA cells were digested for 5 min with the indicated amounts of MNase (milliunits per microgram of DNA). (Far right) Quantification of the data.
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    81
    Boehringer Mannheim micrococcal nuclease mnase
    shRNA-mediated inactivation of ATRX does not alter subtelomeric chromatin accessibility to <t>MNase.</t> (A) Chromatin isolated from 8-MG-BA glioma cells in which ATRX had been inactivated (shATRX) or not (shscrambled [shSCR]) was digested with MNase for the indicated times. (Left gel) Ethidium bromide (EtBr) staining of bulk chromatin. (Right gel) Southern blot with subtelomeric probe. (Far right) Quantification of the data. The signals obtained for mononucleosomes were normalized to the total signals measured for each time point (EtBr or Southern blot). (B) Chromatin samples from shATRX or shSCR 8-MG-BA cells were digested for 5 min with the indicated amounts of MNase (milliunits per microgram of DNA). (Far right) Quantification of the data.
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    Image Search Results


    AGO2 knock-down affects nucleosome occupancy at TSSs bound by SWI/SNF. ( a ) HeLaS3 cells were transfected with a control siRNA (siCTRL) or a pool of AGO2 siRNA (siAGO2). Down-regulation of AGO2 protein was verified by western blot. GAPDH was used as loading control. ( b ) Chromatin from siCTRL- or siAGO2-treated HeLaS3 cells was digested by MNase and recovered DNA fragments were sequenced. Nucleosome occupancy profile for siCTRL and siAGO2 cells was plotted for TSSs with at least 30 swiRNAs (siCTRL, black line; siAGO2, green line). The occupancy at the nucleosome +1 (arrow) is reduced in AGO2 knock-down cells. ( c ) Bars height represents percent reduction of nucleosome occupancy (siAGO2 versus siCTRL) at TSS ±150 nt overlapped by at least the indicated number of swiRNAs (green), IgG-IP ‘other sRNAs’ (black) and AGO1-associated ‘other sRNAs’ (purple). ** P value

    Journal: Nucleic Acids Research

    Article Title: ARGONAUTE2 cooperates with SWI/SNF complex to determine nucleosome occupancy at human Transcription Start Sites

    doi: 10.1093/nar/gku1387

    Figure Lengend Snippet: AGO2 knock-down affects nucleosome occupancy at TSSs bound by SWI/SNF. ( a ) HeLaS3 cells were transfected with a control siRNA (siCTRL) or a pool of AGO2 siRNA (siAGO2). Down-regulation of AGO2 protein was verified by western blot. GAPDH was used as loading control. ( b ) Chromatin from siCTRL- or siAGO2-treated HeLaS3 cells was digested by MNase and recovered DNA fragments were sequenced. Nucleosome occupancy profile for siCTRL and siAGO2 cells was plotted for TSSs with at least 30 swiRNAs (siCTRL, black line; siAGO2, green line). The occupancy at the nucleosome +1 (arrow) is reduced in AGO2 knock-down cells. ( c ) Bars height represents percent reduction of nucleosome occupancy (siAGO2 versus siCTRL) at TSS ±150 nt overlapped by at least the indicated number of swiRNAs (green), IgG-IP ‘other sRNAs’ (black) and AGO1-associated ‘other sRNAs’ (purple). ** P value

    Article Snippet: Isolation of nucleosomal DNA by micrococcal nuclease (MNase) digestion Digestion of chromatin from untreated, siCTRL- or siAGO2-treated HeLa S3 cells (2 × 106 ) was performed with 50 U of MNase (New England Biolabs) in 300 μl of permeabilization buffer (15 mM Tris–HCl pH 7.4, 300 mM sucrose, 60 mM KCl, 15 mM NaCl, 4 mM CaCl2 , 0.5 mM EGTA, 0.2% NP-40, 0.5 mM β-mercaptoethanol) for 20 min at 37°C.

    Techniques: Transfection, Western Blot

    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

    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

    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

    Multiscale analysis of MNase signal. ( a ) Chromosome-wide MNase-Seq coverage along the T. acidophilum chromosome (day2, replicate 2), normalized using sonicated DNA to remove replication-associated coverage bias. ( b ) Multiscale analysis of MNase signal enrichment (see Materials and methods). Significantly enriched or depleted (p-value

    Journal: eLife

    Article Title: The DNA-binding protein HTa from Thermoplasma acidophilum is an archaeal histone analog

    doi: 10.7554/eLife.52542

    Figure Lengend Snippet: Multiscale analysis of MNase signal. ( a ) Chromosome-wide MNase-Seq coverage along the T. acidophilum chromosome (day2, replicate 2), normalized using sonicated DNA to remove replication-associated coverage bias. ( b ) Multiscale analysis of MNase signal enrichment (see Materials and methods). Significantly enriched or depleted (p-value

    Article Snippet: MNase digestion was done on 5 µg of DNA (measured by nanodrop on the reconstituted chromatin) as described above, but omitting the RNAse step.

    Techniques: Sonication, Significance Assay

    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

    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

    Phased nucleosome arrays flanked TSSs of rice genes. (A) Nucleosome positioning profile associated with active genes. Phased nucleosome arrays are detectable after the TSSs. (B) Nucleosome positioning profile associated with non-expressed genes. Phased nucleosome arrays are detected on either side of the TSSs. (C) Distribution of DHS length for five different DHS categories. Note: the length of DHSs associated with proximal promoters (black line) are more variable than the lengths of other DHSs. (D) Heatmap of nucleosome positioning associated with active genes. Left panel: All expressed genes were sorted by the length of DHSs located in proximal promoters. The 5′ ends of the MNase-seq reads were mapped within 1 kb upstream and 1 kb downstream of the TSS of each gene to show the boundaries of nucleosomes core and linker. The red line on the left heatmap indicates the boundaries of DHSs. With the same order of the genes as in the left panel, the 5′ ends of DNase-seq reads (middle panel) and the fragments per kilobase of exon per million fragments mapped (FPKM) value log10 transformation (right panel) were mapped to show the DNase I sensitivity and the expression level of each gene, respectively.

    Journal: PLoS Genetics

    Article Title: Genome-Wide Nucleosome Positioning Is Orchestrated by Genomic Regions Associated with DNase I Hypersensitivity in Rice

    doi: 10.1371/journal.pgen.1004378

    Figure Lengend Snippet: Phased nucleosome arrays flanked TSSs of rice genes. (A) Nucleosome positioning profile associated with active genes. Phased nucleosome arrays are detectable after the TSSs. (B) Nucleosome positioning profile associated with non-expressed genes. Phased nucleosome arrays are detected on either side of the TSSs. (C) Distribution of DHS length for five different DHS categories. Note: the length of DHSs associated with proximal promoters (black line) are more variable than the lengths of other DHSs. (D) Heatmap of nucleosome positioning associated with active genes. Left panel: All expressed genes were sorted by the length of DHSs located in proximal promoters. The 5′ ends of the MNase-seq reads were mapped within 1 kb upstream and 1 kb downstream of the TSS of each gene to show the boundaries of nucleosomes core and linker. The red line on the left heatmap indicates the boundaries of DHSs. With the same order of the genes as in the left panel, the 5′ ends of DNase-seq reads (middle panel) and the fragments per kilobase of exon per million fragments mapped (FPKM) value log10 transformation (right panel) were mapped to show the DNase I sensitivity and the expression level of each gene, respectively.

    Article Snippet: Since the position of the nucleosome center (dyad), which can be identified as the middle position of each paired-end read, is not affected by different levels of MNase digestion, we can calculate the spacing of between two adjacent nucleosomes using the midway point between paired MNase-seq reads rather than 5' ends.

    Techniques: Transformation Assay, Expressing

    Nucleosome positioning profiles associated with DHSs with different lengths in proximal promoters. (A) DHSs in 320–480 bp. (B) DHSs in 200–320 bp. (C) DHSs in 140–200 bp. (D) DHSs in 80–140 bp. (E) DHSs in 20–80 bp. Y-axes show normalized reads of DNase-seq and MNase-seq. Zero on the X-axis indicates the boundary of DHSs toward short arm of the chromosomes. Black ellipses indicate the inferred nucleosomes. Grey ellipses indicate -1 nucleosomes within DHSs. Black vertical lines in (d, e) indicate the left and right boundaries of the DHSs inferred by DNase-seq reads.

    Journal: PLoS Genetics

    Article Title: Genome-Wide Nucleosome Positioning Is Orchestrated by Genomic Regions Associated with DNase I Hypersensitivity in Rice

    doi: 10.1371/journal.pgen.1004378

    Figure Lengend Snippet: Nucleosome positioning profiles associated with DHSs with different lengths in proximal promoters. (A) DHSs in 320–480 bp. (B) DHSs in 200–320 bp. (C) DHSs in 140–200 bp. (D) DHSs in 80–140 bp. (E) DHSs in 20–80 bp. Y-axes show normalized reads of DNase-seq and MNase-seq. Zero on the X-axis indicates the boundary of DHSs toward short arm of the chromosomes. Black ellipses indicate the inferred nucleosomes. Grey ellipses indicate -1 nucleosomes within DHSs. Black vertical lines in (d, e) indicate the left and right boundaries of the DHSs inferred by DNase-seq reads.

    Article Snippet: Since the position of the nucleosome center (dyad), which can be identified as the middle position of each paired-end read, is not affected by different levels of MNase digestion, we can calculate the spacing of between two adjacent nucleosomes using the midway point between paired MNase-seq reads rather than 5' ends.

    Techniques:

    Patterns of nucleosome positioning around DHSs in the rice genome. The nucleosome positioning profiles were shown around the DHSs located in (A) proximal promoters (within 200 bp upstream of a TSS); (B) distal promoters (200–1000 bp upstream of a TSS); (C) within genes; (D) downstream regions of genes (within 200 bp downstream of gene transcription); (E) intergenic regions and (F) 10,000 randomly selected genomic regions. Y-axes show normalized reads (read number in per bp genome in per million reads) within 1 kb upstream and downstream around the DHSs. Ellipses indicate the nucleosomes within (grey) and outside (black) of DHSs. Arrows in (A–D) indicate the direction of gene transcription. Single-end MNase-seq reads were used in mapping nucleosome positioning.

    Journal: PLoS Genetics

    Article Title: Genome-Wide Nucleosome Positioning Is Orchestrated by Genomic Regions Associated with DNase I Hypersensitivity in Rice

    doi: 10.1371/journal.pgen.1004378

    Figure Lengend Snippet: Patterns of nucleosome positioning around DHSs in the rice genome. The nucleosome positioning profiles were shown around the DHSs located in (A) proximal promoters (within 200 bp upstream of a TSS); (B) distal promoters (200–1000 bp upstream of a TSS); (C) within genes; (D) downstream regions of genes (within 200 bp downstream of gene transcription); (E) intergenic regions and (F) 10,000 randomly selected genomic regions. Y-axes show normalized reads (read number in per bp genome in per million reads) within 1 kb upstream and downstream around the DHSs. Ellipses indicate the nucleosomes within (grey) and outside (black) of DHSs. Arrows in (A–D) indicate the direction of gene transcription. Single-end MNase-seq reads were used in mapping nucleosome positioning.

    Article Snippet: Since the position of the nucleosome center (dyad), which can be identified as the middle position of each paired-end read, is not affected by different levels of MNase digestion, we can calculate the spacing of between two adjacent nucleosomes using the midway point between paired MNase-seq reads rather than 5' ends.

    Techniques:

    Patterns of nucleosome positioning around DHSs in the human genome. DHSs (data from CD4+ T cell line) were also divided into five different categories based on their genomic locations: (A) proximal promoters (within 200 bp upstream of a TSS); (B) within genes; and (C) intergenic regions. Y-axes show normalized MNase-seq reads (read number in per bp genome in per million reads). Zero on the x-axes indicates the most sensitive site of the aligned DHSs. Ellipses indicate phased nucleosomes with H2A.Z. Arrows in (A, B) indicate the direction of gene transcription.

    Journal: PLoS Genetics

    Article Title: Genome-Wide Nucleosome Positioning Is Orchestrated by Genomic Regions Associated with DNase I Hypersensitivity in Rice

    doi: 10.1371/journal.pgen.1004378

    Figure Lengend Snippet: Patterns of nucleosome positioning around DHSs in the human genome. DHSs (data from CD4+ T cell line) were also divided into five different categories based on their genomic locations: (A) proximal promoters (within 200 bp upstream of a TSS); (B) within genes; and (C) intergenic regions. Y-axes show normalized MNase-seq reads (read number in per bp genome in per million reads). Zero on the x-axes indicates the most sensitive site of the aligned DHSs. Ellipses indicate phased nucleosomes with H2A.Z. Arrows in (A, B) indicate the direction of gene transcription.

    Article Snippet: Since the position of the nucleosome center (dyad), which can be identified as the middle position of each paired-end read, is not affected by different levels of MNase digestion, we can calculate the spacing of between two adjacent nucleosomes using the midway point between paired MNase-seq reads rather than 5' ends.

    Techniques:

    Association IPA1-binding sites with phased nucleosomes. An example of phased nucleosome arrays that flank an intergenic IPA1-binding site on rice chromosome 8. This binding site is overlapped with a DHS (red arrow). The distribution of MNase-seq data (dyad density calculated from paired-end reads by NucleR) and DNase-seq data (density calculated by F-seq) were used to present the nucleosome and DHS positions. Phased nucleosomes and DHS regions were also schematically marked.

    Journal: PLoS Genetics

    Article Title: Genome-Wide Nucleosome Positioning Is Orchestrated by Genomic Regions Associated with DNase I Hypersensitivity in Rice

    doi: 10.1371/journal.pgen.1004378

    Figure Lengend Snippet: Association IPA1-binding sites with phased nucleosomes. An example of phased nucleosome arrays that flank an intergenic IPA1-binding site on rice chromosome 8. This binding site is overlapped with a DHS (red arrow). The distribution of MNase-seq data (dyad density calculated from paired-end reads by NucleR) and DNase-seq data (density calculated by F-seq) were used to present the nucleosome and DHS positions. Phased nucleosomes and DHS regions were also schematically marked.

    Article Snippet: Since the position of the nucleosome center (dyad), which can be identified as the middle position of each paired-end read, is not affected by different levels of MNase digestion, we can calculate the spacing of between two adjacent nucleosomes using the midway point between paired MNase-seq reads rather than 5' ends.

    Techniques: Binding Assay

    Nucleosome positioning at a (CAG) 85 repeat is not altered in the absence of H2A.1 or H2A.2. A ) Indirect end-labeling of nucleosomal DNA upstream of the CAG repeat. MNase (0, 0.25, 2.5, and 7.5 units) digested DNA was run in 1.5% agarose with ethidium bromide (left) and Southern blotted (right) using a probe ~100 bp proximal to the CAG repeat (red line Figure 1—figure supplement 1A ). Ovals represent nucleosome positions. The experiment was repeated six times; a representative blot is shown. ( B ) Illumina array mapping of nucleosome protection at the CAG repeat. Mononucleosomal DNA from strains containing the (CAG) 85 repeats was hybridized to a custom array of 30-mer probes spanning 425 bp upstream of the repeat to 436 bp downstream of the repeat in YAC CF1. Probes 14–16 contain CAG repeats; probe 15 is composed purely of CAG repeats (probe sequences in Supplementary file 3 ). Error bars represent standard deviation of 2–3 independent experiments.

    Journal: eLife

    Article Title: Distinct roles for S. cerevisiae H2A copies in recombination and repeat stability, with a role for H2A.1 threonine 126

    doi: 10.7554/eLife.53362

    Figure Lengend Snippet: Nucleosome positioning at a (CAG) 85 repeat is not altered in the absence of H2A.1 or H2A.2. A ) Indirect end-labeling of nucleosomal DNA upstream of the CAG repeat. MNase (0, 0.25, 2.5, and 7.5 units) digested DNA was run in 1.5% agarose with ethidium bromide (left) and Southern blotted (right) using a probe ~100 bp proximal to the CAG repeat (red line Figure 1—figure supplement 1A ). Ovals represent nucleosome positions. The experiment was repeated six times; a representative blot is shown. ( B ) Illumina array mapping of nucleosome protection at the CAG repeat. Mononucleosomal DNA from strains containing the (CAG) 85 repeats was hybridized to a custom array of 30-mer probes spanning 425 bp upstream of the repeat to 436 bp downstream of the repeat in YAC CF1. Probes 14–16 contain CAG repeats; probe 15 is composed purely of CAG repeats (probe sequences in Supplementary file 3 ). Error bars represent standard deviation of 2–3 independent experiments.

    Article Snippet: Southern Detection: MNase digested DNA (20–30 μg) was run in 1.5% agarose at 80V for 6 hr and Southern blotted as previously described ( ).

    Techniques: End Labeling, Standard Deviation

    Nucleosome reassembly at the TSS is the initiating event in MLH1 resilencing. A , Regions assayed for nucleosome occupancy using MNase-qPCR (Regions I–IX) and NOMe-Seq (Regions N1 and N2). Shown beneath the gene schematic is NOMe-Seq data from untreated RKO cells at Region N1. Black arrows indicate the MLH1 and EPM2AIP1 TSS. Bottom panel represents GpC accessibility. Black circles = GpC dinucleotides methylated/accessible to the GpC methyltransferase M. Cvi PI. White circles = GpC dinucleotides not methylated/inaccessible to GpC methyltransferase. Pink shading indicates regions of inaccessibility of ≥150 bp. Asterix = region of M. Cvi PI accessibility. B , Relative nucleosome levels in untreated RKO cells at the indicated regions (black bars labeled Regions I–IX) as determined by MNase-qPCR. Drawn to scale with schematic shown in A. Error bars = SD. C and D , qPCR results showing changes in relative nucleosome levels at Regions III and VI following decitabine exposure. E and F , MLH1 gene expression (E) and promoter bisulfite pyrosequencing (F), reproduced from Figure 2A and D for ease of comparison with nucleosome levels. G–I , NOMe-Seq analysis of the MLH1 promoter at Region N2 in SW620 (F) and RKO (G,H) cells at the indicated treatment points. Black filled triangles = methylated CpG dinucleotides; white filled triangles = unmethylated CpG dinucleotides. See also Figure S2 .

    Journal: PLoS Genetics

    Article Title: Reassembly of Nucleosomes at the MLH1 Promoter Initiates Resilencing Following Decitabine Exposure

    doi: 10.1371/journal.pgen.1003636

    Figure Lengend Snippet: Nucleosome reassembly at the TSS is the initiating event in MLH1 resilencing. A , Regions assayed for nucleosome occupancy using MNase-qPCR (Regions I–IX) and NOMe-Seq (Regions N1 and N2). Shown beneath the gene schematic is NOMe-Seq data from untreated RKO cells at Region N1. Black arrows indicate the MLH1 and EPM2AIP1 TSS. Bottom panel represents GpC accessibility. Black circles = GpC dinucleotides methylated/accessible to the GpC methyltransferase M. Cvi PI. White circles = GpC dinucleotides not methylated/inaccessible to GpC methyltransferase. Pink shading indicates regions of inaccessibility of ≥150 bp. Asterix = region of M. Cvi PI accessibility. B , Relative nucleosome levels in untreated RKO cells at the indicated regions (black bars labeled Regions I–IX) as determined by MNase-qPCR. Drawn to scale with schematic shown in A. Error bars = SD. C and D , qPCR results showing changes in relative nucleosome levels at Regions III and VI following decitabine exposure. E and F , MLH1 gene expression (E) and promoter bisulfite pyrosequencing (F), reproduced from Figure 2A and D for ease of comparison with nucleosome levels. G–I , NOMe-Seq analysis of the MLH1 promoter at Region N2 in SW620 (F) and RKO (G,H) cells at the indicated treatment points. Black filled triangles = methylated CpG dinucleotides; white filled triangles = unmethylated CpG dinucleotides. See also Figure S2 .

    Article Snippet: Isolation of mononucleosome DNA using micrococcal nuclease (MNase) and qPCR A total of 1×107 cells were lysed on ice in 50 mM Tris HCl pH 7.9, 100 mM KCl, 5 mM MgCl2 , 50% (v/v) glycerol, 1.5% (v/v) β-mercaptoethanol, 0.1% (w/v) Saponin and Complete Protease Inhibitor with EDTA (Roche) followed by equilibration in 50 mM Tris HCl pH 7.5, 0.32 mM sucrose, 4 mM MgCl2 , 1 mM CaCl2 , and EDTA-free Complete Protease Inhibitors (Roche).

    Techniques: Real-time Polymerase Chain Reaction, Gel Permeation Chromatography, Methylation, Labeling, Expressing

    shRNA-mediated inactivation of ATRX does not alter subtelomeric chromatin accessibility to MNase. (A) Chromatin isolated from 8-MG-BA glioma cells in which ATRX had been inactivated (shATRX) or not (shscrambled [shSCR]) was digested with MNase for the indicated times. (Left gel) Ethidium bromide (EtBr) staining of bulk chromatin. (Right gel) Southern blot with subtelomeric probe. (Far right) Quantification of the data. The signals obtained for mononucleosomes were normalized to the total signals measured for each time point (EtBr or Southern blot). (B) Chromatin samples from shATRX or shSCR 8-MG-BA cells were digested for 5 min with the indicated amounts of MNase (milliunits per microgram of DNA). (Far right) Quantification of the data.

    Journal: Molecular and Cellular Biology

    Article Title: Genetic Inactivation of ATRX Leads to a Decrease in the Amount of Telomeric Cohesin and Level of Telomere Transcription in Human Glioma Cells

    doi: 10.1128/MCB.01317-14

    Figure Lengend Snippet: shRNA-mediated inactivation of ATRX does not alter subtelomeric chromatin accessibility to MNase. (A) Chromatin isolated from 8-MG-BA glioma cells in which ATRX had been inactivated (shATRX) or not (shscrambled [shSCR]) was digested with MNase for the indicated times. (Left gel) Ethidium bromide (EtBr) staining of bulk chromatin. (Right gel) Southern blot with subtelomeric probe. (Far right) Quantification of the data. The signals obtained for mononucleosomes were normalized to the total signals measured for each time point (EtBr or Southern blot). (B) Chromatin samples from shATRX or shSCR 8-MG-BA cells were digested for 5 min with the indicated amounts of MNase (milliunits per microgram of DNA). (Far right) Quantification of the data.

    Article Snippet: Nuclei isolated from 107 cells were digested with 8 mU of micrococcal nuclease (MNase) (Sigma)/μg DNA at 37°C for the indicated times, as described previously ( ).

    Techniques: shRNA, Isolation, Staining, Southern Blot