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  • 95
    New England Biolabs micrococcal nuclease mnase digestion digestion
    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
    Micrococcal Nuclease Mnase Digestion Digestion, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Worthington Biochemical micrococcal nuclease
    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
    Micrococcal Nuclease, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 95/100, based on 913 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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.
    Micrococcal Nuclease Mnase, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa micrococcal nuclease
    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.
    Micrococcal Nuclease, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 236 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche nuclease s7 micrococcal nuclease mnase roche
    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.
    Nuclease S7 Micrococcal Nuclease Mnase Roche, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc micrococcal nuclease mnase
    Genome-wide nucleosome positioning in Dictyostelium . ( A ) Normalized read midpoint frequency distributions of <t>MNase-protected</t> fragments (nucleosome dyads) of all 12,750 genes in growth-stage WT cells were aligned relative to their ATG codons. Peaks (arrows) correspond to dyad midpoints for globally phased nucleosomes in the 5′ region of intragenic <t>DNA,</t> and distances between mapped read peaks correspond to ∼170 bp NRL. The protein coding DNA sequence (cds) region is shaded. ( B ) Normalized read midpoint frequency distributions of all genes in growth-stage WT cells were aligned relative to their translational termination sites (stop codons). Peaks (arrows) in the mean normalized frequency distribution correspond to globally phased nucleosomes in the 3′ region of intragenic DNA. The protein cds region is shaded. ( C ) Normalized dyad read midpoint frequency distributions for WT chromatin (CHR; dotted line) (see A ) were adjusted for sequence mappability by dividing with equivalent control data from MNase-digested naked (protein free) WT DNA (DNA; red line) and replotted as the ratio (CHR/DNA; thick black line) within 1.2 kb of flanking chromatin relative to ATG sites of all 12,750 genes. An ∼170-bp nucleosome-depleted (“free”) region (NDR) is centered near the AT-rich regions of Dictyostelium TSS. Positioned nucleosomes upstream (+) and downstream (−) to the NDR are indicated by arrows. The protein cds region is shaded.
    Micrococcal Nuclease Mnase, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 90/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche micrococcal nuclease mnase
    Genome-wide nucleosome positioning in Dictyostelium . ( A ) Normalized read midpoint frequency distributions of <t>MNase-protected</t> fragments (nucleosome dyads) of all 12,750 genes in growth-stage WT cells were aligned relative to their ATG codons. Peaks (arrows) correspond to dyad midpoints for globally phased nucleosomes in the 5′ region of intragenic <t>DNA,</t> and distances between mapped read peaks correspond to ∼170 bp NRL. The protein coding DNA sequence (cds) region is shaded. ( B ) Normalized read midpoint frequency distributions of all genes in growth-stage WT cells were aligned relative to their translational termination sites (stop codons). Peaks (arrows) in the mean normalized frequency distribution correspond to globally phased nucleosomes in the 3′ region of intragenic DNA. The protein cds region is shaded. ( C ) Normalized dyad read midpoint frequency distributions for WT chromatin (CHR; dotted line) (see A ) were adjusted for sequence mappability by dividing with equivalent control data from MNase-digested naked (protein free) WT DNA (DNA; red line) and replotted as the ratio (CHR/DNA; thick black line) within 1.2 kb of flanking chromatin relative to ATG sites of all 12,750 genes. An ∼170-bp nucleosome-depleted (“free”) region (NDR) is centered near the AT-rich regions of Dictyostelium TSS. Positioned nucleosomes upstream (+) and downstream (−) to the NDR are indicated by arrows. The protein cds region is shaded.
    Micrococcal Nuclease Mnase, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 109 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mnase  (Roche)
    99
    Roche 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.
    Mnase, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 244 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mnase
    Neutrophils release calprotectin by forming NETs. (A–F) Confocal images of human neutrophils without stimulation (A), after 0.5 h (B), 1 h (C), 2 h (D), 3 h (E) and 4 h (F) after activation. Samples were stained with antibodies specific for the calprotectin heteroduplex (red) and for MPO (green). <t>DNA</t> was stained with DRAQ5 (blue). Calprotectin localizes to the cytoplasm and partially to the nucleus (A, arrow). After stimulation for 0.5 h (B) the neutrophils flattened and formed numerous vacuoles. This reveals a granular staining for MPO and a more dispersed cytoplasmic staining for calprotectin. After stimulation for 1 h (C) the neutrophils round up slightly. The MPO and calprotectin stain partially overlap in the cytoplasm. After stimulation for 2 h (D), calprotectin, MPO and nuclear DNA start to colocalize in the decondensed nucleus (purple). After 3 h (E) and more so after 4 h (F) of stimulation, the cell membrane ruptures and calprotectin is released in NETs colocalizing with MPO and DNA. Scale bar = 10 µm; one experiment out of two is shown. (G–I) Subunits of calprotectin S100A8 and S100A9 are released after cell death during NET formation and not by degranulation. NET formation was induced with PMA and degranulation using formyl-met-leu-phe (f-MLP). (G) Neutrophil death was monitored by quantification of LDH activity in supernatants calculated as means±s.d. (n = 3). (H) Release of S100A8, S100A9, lactotransferrin (LTF) and myeloperoxidase (MPO) were analyzed by immunoblotting. one experiment out of two is shown. (I) Quantification of immunoblots using 2D densitometry analyzing S100A9 protein preparations from supernatants (lane 1), <t>MNase-digested</t> NETs (lane 2) and cell remnants indigestible for MNase (lane 3). Values were calculated as means±s.d. (n = 3) from one experiment out of two.
    Mnase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 417 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa mnase
    Neutrophils release calprotectin by forming NETs. (A–F) Confocal images of human neutrophils without stimulation (A), after 0.5 h (B), 1 h (C), 2 h (D), 3 h (E) and 4 h (F) after activation. Samples were stained with antibodies specific for the calprotectin heteroduplex (red) and for MPO (green). <t>DNA</t> was stained with DRAQ5 (blue). Calprotectin localizes to the cytoplasm and partially to the nucleus (A, arrow). After stimulation for 0.5 h (B) the neutrophils flattened and formed numerous vacuoles. This reveals a granular staining for MPO and a more dispersed cytoplasmic staining for calprotectin. After stimulation for 1 h (C) the neutrophils round up slightly. The MPO and calprotectin stain partially overlap in the cytoplasm. After stimulation for 2 h (D), calprotectin, MPO and nuclear DNA start to colocalize in the decondensed nucleus (purple). After 3 h (E) and more so after 4 h (F) of stimulation, the cell membrane ruptures and calprotectin is released in NETs colocalizing with MPO and DNA. Scale bar = 10 µm; one experiment out of two is shown. (G–I) Subunits of calprotectin S100A8 and S100A9 are released after cell death during NET formation and not by degranulation. NET formation was induced with PMA and degranulation using formyl-met-leu-phe (f-MLP). (G) Neutrophil death was monitored by quantification of LDH activity in supernatants calculated as means±s.d. (n = 3). (H) Release of S100A8, S100A9, lactotransferrin (LTF) and myeloperoxidase (MPO) were analyzed by immunoblotting. one experiment out of two is shown. (I) Quantification of immunoblots using 2D densitometry analyzing S100A9 protein preparations from supernatants (lane 1), <t>MNase-digested</t> NETs (lane 2) and cell remnants indigestible for MNase (lane 3). Values were calculated as means±s.d. (n = 3) from one experiment out of two.
    Mnase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 255 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 855 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc 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 Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Worthington Biochemical microccocal nuclease
    Chromatin-remodeling (nucleosome insertion and rearrangement) on Nanog gene promoter. ( A ) <t>ESCs</t> were treated with RA for 3 days, then treated with 5, 10 and 30 U of <t>MNase</t> for 6 min at 37°C. Extracted chromatin DNA was separated on 1.5% agarose gels followed by Southern blot hybridization with 32 P-labeled 500-bp probes specific to three regions on the Nanog promoter. Positions of probes are depicted on the map. ( B ) Restriction enzyme accessibility of Nanog promoter region in ESCs. Asterisk marks the diagnostic band indicative of each sensitive site. The results are summarized and shown on the map above these blots. Restriction sites are labeled under the map. ( C ) N1, N2, N3 and N4 PCR fragments, amplified from mononucleosomal DNA using the primers a/b (N1), c/d (N2), e/f (N3) and g/h (N4). Primer sequences were listed in Supplementary Table S1 . Genomic DNAs isolated from 1 × 10 6 cells were used for N1 amplification as control (upper panel). ESCs were transfected with siRNA specific for Brm or siRNA control. Changes in mononucleosome formation on the Nanog gene promoter were monitored (bottom panel). ( D ) Upper: nucleosome occupancy on the Nanog promoter in ESCs with or without RA treatment. The gray-highlighted region (q8–12) represents the location of the N2 nucleosome formation. Lower: analysis of nucleosome occupancy at the q8–12 regions of Nanog in control and Brm-knockdown ES cell by the MNase resistance assay. Data points represent average qPCR signals from two independent experiments.
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    Thermo Fisher micrococcal nuclease mnase
    Chromatin-remodeling (nucleosome insertion and rearrangement) on Nanog gene promoter. ( A ) <t>ESCs</t> were treated with RA for 3 days, then treated with 5, 10 and 30 U of <t>MNase</t> for 6 min at 37°C. Extracted chromatin DNA was separated on 1.5% agarose gels followed by Southern blot hybridization with 32 P-labeled 500-bp probes specific to three regions on the Nanog promoter. Positions of probes are depicted on the map. ( B ) Restriction enzyme accessibility of Nanog promoter region in ESCs. Asterisk marks the diagnostic band indicative of each sensitive site. The results are summarized and shown on the map above these blots. Restriction sites are labeled under the map. ( C ) N1, N2, N3 and N4 PCR fragments, amplified from mononucleosomal DNA using the primers a/b (N1), c/d (N2), e/f (N3) and g/h (N4). Primer sequences were listed in Supplementary Table S1 . Genomic DNAs isolated from 1 × 10 6 cells were used for N1 amplification as control (upper panel). ESCs were transfected with siRNA specific for Brm or siRNA control. Changes in mononucleosome formation on the Nanog gene promoter were monitored (bottom panel). ( D ) Upper: nucleosome occupancy on the Nanog promoter in ESCs with or without RA treatment. The gray-highlighted region (q8–12) represents the location of the N2 nucleosome formation. Lower: analysis of nucleosome occupancy at the q8–12 regions of Nanog in control and Brm-knockdown ES cell by the MNase resistance assay. Data points represent average qPCR signals from two independent experiments.
    Micrococcal Nuclease Mnase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    TaKaRa mnase i
    Chromatin-remodeling (nucleosome insertion and rearrangement) on Nanog gene promoter. ( A ) <t>ESCs</t> were treated with RA for 3 days, then treated with 5, 10 and 30 U of <t>MNase</t> for 6 min at 37°C. Extracted chromatin DNA was separated on 1.5% agarose gels followed by Southern blot hybridization with 32 P-labeled 500-bp probes specific to three regions on the Nanog promoter. Positions of probes are depicted on the map. ( B ) Restriction enzyme accessibility of Nanog promoter region in ESCs. Asterisk marks the diagnostic band indicative of each sensitive site. The results are summarized and shown on the map above these blots. Restriction sites are labeled under the map. ( C ) N1, N2, N3 and N4 PCR fragments, amplified from mononucleosomal DNA using the primers a/b (N1), c/d (N2), e/f (N3) and g/h (N4). Primer sequences were listed in Supplementary Table S1 . Genomic DNAs isolated from 1 × 10 6 cells were used for N1 amplification as control (upper panel). ESCs were transfected with siRNA specific for Brm or siRNA control. Changes in mononucleosome formation on the Nanog gene promoter were monitored (bottom panel). ( D ) Upper: nucleosome occupancy on the Nanog promoter in ESCs with or without RA treatment. The gray-highlighted region (q8–12) represents the location of the N2 nucleosome formation. Lower: analysis of nucleosome occupancy at the q8–12 regions of Nanog in control and Brm-knockdown ES cell by the MNase resistance assay. Data points represent average qPCR signals from two independent experiments.
    Mnase I, supplied by TaKaRa, used in various techniques. Bioz Stars score: 83/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Active Motif mnase cocktail
    Chromatin-remodeling (nucleosome insertion and rearrangement) on Nanog gene promoter. ( A ) <t>ESCs</t> were treated with RA for 3 days, then treated with 5, 10 and 30 U of <t>MNase</t> for 6 min at 37°C. Extracted chromatin DNA was separated on 1.5% agarose gels followed by Southern blot hybridization with 32 P-labeled 500-bp probes specific to three regions on the Nanog promoter. Positions of probes are depicted on the map. ( B ) Restriction enzyme accessibility of Nanog promoter region in ESCs. Asterisk marks the diagnostic band indicative of each sensitive site. The results are summarized and shown on the map above these blots. Restriction sites are labeled under the map. ( C ) N1, N2, N3 and N4 PCR fragments, amplified from mononucleosomal DNA using the primers a/b (N1), c/d (N2), e/f (N3) and g/h (N4). Primer sequences were listed in Supplementary Table S1 . Genomic DNAs isolated from 1 × 10 6 cells were used for N1 amplification as control (upper panel). ESCs were transfected with siRNA specific for Brm or siRNA control. Changes in mononucleosome formation on the Nanog gene promoter were monitored (bottom panel). ( D ) Upper: nucleosome occupancy on the Nanog promoter in ESCs with or without RA treatment. The gray-highlighted region (q8–12) represents the location of the N2 nucleosome formation. Lower: analysis of nucleosome occupancy at the q8–12 regions of Nanog in control and Brm-knockdown ES cell by the MNase resistance assay. Data points represent average qPCR signals from two independent experiments.
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    83
    Roche mnase s7
    Chromatin-remodeling (nucleosome insertion and rearrangement) on Nanog gene promoter. ( A ) <t>ESCs</t> were treated with RA for 3 days, then treated with 5, 10 and 30 U of <t>MNase</t> for 6 min at 37°C. Extracted chromatin DNA was separated on 1.5% agarose gels followed by Southern blot hybridization with 32 P-labeled 500-bp probes specific to three regions on the Nanog promoter. Positions of probes are depicted on the map. ( B ) Restriction enzyme accessibility of Nanog promoter region in ESCs. Asterisk marks the diagnostic band indicative of each sensitive site. The results are summarized and shown on the map above these blots. Restriction sites are labeled under the map. ( C ) N1, N2, N3 and N4 PCR fragments, amplified from mononucleosomal DNA using the primers a/b (N1), c/d (N2), e/f (N3) and g/h (N4). Primer sequences were listed in Supplementary Table S1 . Genomic DNAs isolated from 1 × 10 6 cells were used for N1 amplification as control (upper panel). ESCs were transfected with siRNA specific for Brm or siRNA control. Changes in mononucleosome formation on the Nanog gene promoter were monitored (bottom panel). ( D ) Upper: nucleosome occupancy on the Nanog promoter in ESCs with or without RA treatment. The gray-highlighted region (q8–12) represents the location of the N2 nucleosome formation. Lower: analysis of nucleosome occupancy at the q8–12 regions of Nanog in control and Brm-knockdown ES cell by the MNase resistance assay. Data points represent average qPCR signals from two independent experiments.
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    Millipore mnase i
    Chromatin-remodeling (nucleosome insertion and rearrangement) on Nanog gene promoter. ( A ) <t>ESCs</t> were treated with RA for 3 days, then treated with 5, 10 and 30 U of <t>MNase</t> for 6 min at 37°C. Extracted chromatin DNA was separated on 1.5% agarose gels followed by Southern blot hybridization with 32 P-labeled 500-bp probes specific to three regions on the Nanog promoter. Positions of probes are depicted on the map. ( B ) Restriction enzyme accessibility of Nanog promoter region in ESCs. Asterisk marks the diagnostic band indicative of each sensitive site. The results are summarized and shown on the map above these blots. Restriction sites are labeled under the map. ( C ) N1, N2, N3 and N4 PCR fragments, amplified from mononucleosomal DNA using the primers a/b (N1), c/d (N2), e/f (N3) and g/h (N4). Primer sequences were listed in Supplementary Table S1 . Genomic DNAs isolated from 1 × 10 6 cells were used for N1 amplification as control (upper panel). ESCs were transfected with siRNA specific for Brm or siRNA control. Changes in mononucleosome formation on the Nanog gene promoter were monitored (bottom panel). ( D ) Upper: nucleosome occupancy on the Nanog promoter in ESCs with or without RA treatment. The gray-highlighted region (q8–12) represents the location of the N2 nucleosome formation. Lower: analysis of nucleosome occupancy at the q8–12 regions of Nanog in control and Brm-knockdown ES cell by the MNase resistance assay. Data points represent average qPCR signals from two independent experiments.
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    Thermo Fisher mnase digestion buffer
    Chromatin-remodeling (nucleosome insertion and rearrangement) on Nanog gene promoter. ( A ) <t>ESCs</t> were treated with RA for 3 days, then treated with 5, 10 and 30 U of <t>MNase</t> for 6 min at 37°C. Extracted chromatin DNA was separated on 1.5% agarose gels followed by Southern blot hybridization with 32 P-labeled 500-bp probes specific to three regions on the Nanog promoter. Positions of probes are depicted on the map. ( B ) Restriction enzyme accessibility of Nanog promoter region in ESCs. Asterisk marks the diagnostic band indicative of each sensitive site. The results are summarized and shown on the map above these blots. Restriction sites are labeled under the map. ( C ) N1, N2, N3 and N4 PCR fragments, amplified from mononucleosomal DNA using the primers a/b (N1), c/d (N2), e/f (N3) and g/h (N4). Primer sequences were listed in Supplementary Table S1 . Genomic DNAs isolated from 1 × 10 6 cells were used for N1 amplification as control (upper panel). ESCs were transfected with siRNA specific for Brm or siRNA control. Changes in mononucleosome formation on the Nanog gene promoter were monitored (bottom panel). ( D ) Upper: nucleosome occupancy on the Nanog promoter in ESCs with or without RA treatment. The gray-highlighted region (q8–12) represents the location of the N2 nucleosome formation. Lower: analysis of nucleosome occupancy at the q8–12 regions of Nanog in control and Brm-knockdown ES cell by the MNase resistance assay. Data points represent average qPCR signals from two independent experiments.
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    Millipore 25 u microccocal nuclease
    Chromatin-remodeling (nucleosome insertion and rearrangement) on Nanog gene promoter. ( A ) <t>ESCs</t> were treated with RA for 3 days, then treated with 5, 10 and 30 U of <t>MNase</t> for 6 min at 37°C. Extracted chromatin DNA was separated on 1.5% agarose gels followed by Southern blot hybridization with 32 P-labeled 500-bp probes specific to three regions on the Nanog promoter. Positions of probes are depicted on the map. ( B ) Restriction enzyme accessibility of Nanog promoter region in ESCs. Asterisk marks the diagnostic band indicative of each sensitive site. The results are summarized and shown on the map above these blots. Restriction sites are labeled under the map. ( C ) N1, N2, N3 and N4 PCR fragments, amplified from mononucleosomal DNA using the primers a/b (N1), c/d (N2), e/f (N3) and g/h (N4). Primer sequences were listed in Supplementary Table S1 . Genomic DNAs isolated from 1 × 10 6 cells were used for N1 amplification as control (upper panel). ESCs were transfected with siRNA specific for Brm or siRNA control. Changes in mononucleosome formation on the Nanog gene promoter were monitored (bottom panel). ( D ) Upper: nucleosome occupancy on the Nanog promoter in ESCs with or without RA treatment. The gray-highlighted region (q8–12) represents the location of the N2 nucleosome formation. Lower: analysis of nucleosome occupancy at the q8–12 regions of Nanog in control and Brm-knockdown ES cell by the MNase resistance assay. Data points represent average qPCR signals from two independent experiments.
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    New England Biolabs mnase
    Differential chromatin structure and transcription factor binding between consensus and Alu RBPJ binding sites. ( A ) Heat map of clustered reads densities for the indicated genome-wide determination or <t>DNA</t> sequence feature centered around RBPJ peak summits for all 28 220 RBPJ binding sites. ( B ) The same analysis as in A for the 4921 RBPJ peak summits that intersected with an Alu element within 200 bp. ( C ) Quantification of DNA ends densities for <t>MNAse</t> digested input, RBPJ immunoprecipitated, and DNase hypersensitive DNA for cluster 1 (without Alu) and cluster 5 (with Alu) regions.
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    Diagenode mnase
    Differential chromatin structure and transcription factor binding between consensus and Alu RBPJ binding sites. ( A ) Heat map of clustered reads densities for the indicated genome-wide determination or <t>DNA</t> sequence feature centered around RBPJ peak summits for all 28 220 RBPJ binding sites. ( B ) The same analysis as in A for the 4921 RBPJ peak summits that intersected with an Alu element within 200 bp. ( C ) Quantification of DNA ends densities for <t>MNAse</t> digested input, RBPJ immunoprecipitated, and DNase hypersensitive DNA for cluster 1 (without Alu) and cluster 5 (with Alu) regions.
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    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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    Illumina Inc mnase digested chromatin
    Fun30 is required for normal CEN -flanking nucleosome positioning and/or CEN core particle structure. A) Genome browser trace of Fun30 ChIP enrichment and nucleosome dyad frequency centred on and surrounding yeast CEN1 . The upper trace shows Log 2 Fun30 ChIP-seq enrichment values binned at 10 bp intervals and smoothed with a 3 bin moving average. Wildtype (WT) and Δfun30 chromatin was digested with <t>MNase</t> and nuclease-protected DNA species sequenced using paired-end mode <t>Illumina</t> technology. Nucleosome sequencing data (nuc) traces were plotted as mirror images in the lower panel. The graph shows a map of the centre point positions of paired sequence reads with end-to-end distances of 150 bp+/−20% wild-type and Δfun30 mutant chromatin samples surrounding CEN1 . The frequency distributions, which effectively map chromatin particle dyads, were binned at 10 bp intervals, and smoothed by applying a 3 bin moving average. Peaks in the dyad distributions correspond to translationally-positioned nucleosomes in the original genome. The CEN core particle is also mapped using this method and can be visualised as a small peak centred on the CEN region marked with a grey box. Pink bars show the positions of ORFs (B–D) Genome browser plots of Fun30 ChIP-seq and nucleosome sequence distributions as described above for CEN10, 11 and 12 respectively. Fun30-dependent changes in the height of a nucleosome dyad or CEN core particle peak are marked with a red asterix. Fun30-dependent changes in the position of a CEN -flanking nucleosome dyad peak are marked with red arrows. Genome browser plots for all yeast CENs are shown in Figure S6 .
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    Solexa mnase digestion
    Fun30 is required for normal CEN -flanking nucleosome positioning and/or CEN core particle structure. A) Genome browser trace of Fun30 ChIP enrichment and nucleosome dyad frequency centred on and surrounding yeast CEN1 . The upper trace shows Log 2 Fun30 ChIP-seq enrichment values binned at 10 bp intervals and smoothed with a 3 bin moving average. Wildtype (WT) and Δfun30 chromatin was digested with <t>MNase</t> and nuclease-protected DNA species sequenced using paired-end mode <t>Illumina</t> technology. Nucleosome sequencing data (nuc) traces were plotted as mirror images in the lower panel. The graph shows a map of the centre point positions of paired sequence reads with end-to-end distances of 150 bp+/−20% wild-type and Δfun30 mutant chromatin samples surrounding CEN1 . The frequency distributions, which effectively map chromatin particle dyads, were binned at 10 bp intervals, and smoothed by applying a 3 bin moving average. Peaks in the dyad distributions correspond to translationally-positioned nucleosomes in the original genome. The CEN core particle is also mapped using this method and can be visualised as a small peak centred on the CEN region marked with a grey box. Pink bars show the positions of ORFs (B–D) Genome browser plots of Fun30 ChIP-seq and nucleosome sequence distributions as described above for CEN10, 11 and 12 respectively. Fun30-dependent changes in the height of a nucleosome dyad or CEN core particle peak are marked with a red asterix. Fun30-dependent changes in the position of a CEN -flanking nucleosome dyad peak are marked with red arrows. Genome browser plots for all yeast CENs are shown in Figure S6 .
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    Cell Signaling Technology Inc micrococcal nuclease
    Fun30 is required for normal CEN -flanking nucleosome positioning and/or CEN core particle structure. A) Genome browser trace of Fun30 ChIP enrichment and nucleosome dyad frequency centred on and surrounding yeast CEN1 . The upper trace shows Log 2 Fun30 ChIP-seq enrichment values binned at 10 bp intervals and smoothed with a 3 bin moving average. Wildtype (WT) and Δfun30 chromatin was digested with <t>MNase</t> and nuclease-protected DNA species sequenced using paired-end mode <t>Illumina</t> technology. Nucleosome sequencing data (nuc) traces were plotted as mirror images in the lower panel. The graph shows a map of the centre point positions of paired sequence reads with end-to-end distances of 150 bp+/−20% wild-type and Δfun30 mutant chromatin samples surrounding CEN1 . The frequency distributions, which effectively map chromatin particle dyads, were binned at 10 bp intervals, and smoothed by applying a 3 bin moving average. Peaks in the dyad distributions correspond to translationally-positioned nucleosomes in the original genome. The CEN core particle is also mapped using this method and can be visualised as a small peak centred on the CEN region marked with a grey box. Pink bars show the positions of ORFs (B–D) Genome browser plots of Fun30 ChIP-seq and nucleosome sequence distributions as described above for CEN10, 11 and 12 respectively. Fun30-dependent changes in the height of a nucleosome dyad or CEN core particle peak are marked with a red asterix. Fun30-dependent changes in the position of a CEN -flanking nucleosome dyad peak are marked with red arrows. Genome browser plots for all yeast CENs are shown in Figure S6 .
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    New England Biolabs mnase i
    Fun30 is required for normal CEN -flanking nucleosome positioning and/or CEN core particle structure. A) Genome browser trace of Fun30 ChIP enrichment and nucleosome dyad frequency centred on and surrounding yeast CEN1 . The upper trace shows Log 2 Fun30 ChIP-seq enrichment values binned at 10 bp intervals and smoothed with a 3 bin moving average. Wildtype (WT) and Δfun30 chromatin was digested with <t>MNase</t> and nuclease-protected DNA species sequenced using paired-end mode <t>Illumina</t> technology. Nucleosome sequencing data (nuc) traces were plotted as mirror images in the lower panel. The graph shows a map of the centre point positions of paired sequence reads with end-to-end distances of 150 bp+/−20% wild-type and Δfun30 mutant chromatin samples surrounding CEN1 . The frequency distributions, which effectively map chromatin particle dyads, were binned at 10 bp intervals, and smoothed by applying a 3 bin moving average. Peaks in the dyad distributions correspond to translationally-positioned nucleosomes in the original genome. The CEN core particle is also mapped using this method and can be visualised as a small peak centred on the CEN region marked with a grey box. Pink bars show the positions of ORFs (B–D) Genome browser plots of Fun30 ChIP-seq and nucleosome sequence distributions as described above for CEN10, 11 and 12 respectively. Fun30-dependent changes in the height of a nucleosome dyad or CEN core particle peak are marked with a red asterix. Fun30-dependent changes in the position of a CEN -flanking nucleosome dyad peak are marked with red arrows. Genome browser plots for all yeast CENs are shown in Figure S6 .
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    Worthington Biochemical mnase i
    Fun30 is required for normal CEN -flanking nucleosome positioning and/or CEN core particle structure. A) Genome browser trace of Fun30 ChIP enrichment and nucleosome dyad frequency centred on and surrounding yeast CEN1 . The upper trace shows Log 2 Fun30 ChIP-seq enrichment values binned at 10 bp intervals and smoothed with a 3 bin moving average. Wildtype (WT) and Δfun30 chromatin was digested with <t>MNase</t> and nuclease-protected DNA species sequenced using paired-end mode <t>Illumina</t> technology. Nucleosome sequencing data (nuc) traces were plotted as mirror images in the lower panel. The graph shows a map of the centre point positions of paired sequence reads with end-to-end distances of 150 bp+/−20% wild-type and Δfun30 mutant chromatin samples surrounding CEN1 . The frequency distributions, which effectively map chromatin particle dyads, were binned at 10 bp intervals, and smoothed by applying a 3 bin moving average. Peaks in the dyad distributions correspond to translationally-positioned nucleosomes in the original genome. The CEN core particle is also mapped using this method and can be visualised as a small peak centred on the CEN region marked with a grey box. Pink bars show the positions of ORFs (B–D) Genome browser plots of Fun30 ChIP-seq and nucleosome sequence distributions as described above for CEN10, 11 and 12 respectively. Fun30-dependent changes in the height of a nucleosome dyad or CEN core particle peak are marked with a red asterix. Fun30-dependent changes in the position of a CEN -flanking nucleosome dyad peak are marked with red arrows. Genome browser plots for all yeast CENs are shown in Figure S6 .
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    Zymo Research ez nucleosomal dna kit
    Fun30 is required for normal CEN -flanking nucleosome positioning and/or CEN core particle structure. A) Genome browser trace of Fun30 ChIP enrichment and nucleosome dyad frequency centred on and surrounding yeast CEN1 . The upper trace shows Log 2 Fun30 ChIP-seq enrichment values binned at 10 bp intervals and smoothed with a 3 bin moving average. Wildtype (WT) and Δfun30 chromatin was digested with <t>MNase</t> and nuclease-protected DNA species sequenced using paired-end mode <t>Illumina</t> technology. Nucleosome sequencing data (nuc) traces were plotted as mirror images in the lower panel. The graph shows a map of the centre point positions of paired sequence reads with end-to-end distances of 150 bp+/−20% wild-type and Δfun30 mutant chromatin samples surrounding CEN1 . The frequency distributions, which effectively map chromatin particle dyads, were binned at 10 bp intervals, and smoothed by applying a 3 bin moving average. Peaks in the dyad distributions correspond to translationally-positioned nucleosomes in the original genome. The CEN core particle is also mapped using this method and can be visualised as a small peak centred on the CEN region marked with a grey box. Pink bars show the positions of ORFs (B–D) Genome browser plots of Fun30 ChIP-seq and nucleosome sequence distributions as described above for CEN10, 11 and 12 respectively. Fun30-dependent changes in the height of a nucleosome dyad or CEN core particle peak are marked with a red asterix. Fun30-dependent changes in the position of a CEN -flanking nucleosome dyad peak are marked with red arrows. Genome browser plots for all yeast CENs are shown in Figure S6 .
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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

    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

    Genome-wide nucleosome positioning in Dictyostelium . ( A ) Normalized read midpoint frequency distributions of MNase-protected fragments (nucleosome dyads) of all 12,750 genes in growth-stage WT cells were aligned relative to their ATG codons. Peaks (arrows) correspond to dyad midpoints for globally phased nucleosomes in the 5′ region of intragenic DNA, and distances between mapped read peaks correspond to ∼170 bp NRL. The protein coding DNA sequence (cds) region is shaded. ( B ) Normalized read midpoint frequency distributions of all genes in growth-stage WT cells were aligned relative to their translational termination sites (stop codons). Peaks (arrows) in the mean normalized frequency distribution correspond to globally phased nucleosomes in the 3′ region of intragenic DNA. The protein cds region is shaded. ( C ) Normalized dyad read midpoint frequency distributions for WT chromatin (CHR; dotted line) (see A ) were adjusted for sequence mappability by dividing with equivalent control data from MNase-digested naked (protein free) WT DNA (DNA; red line) and replotted as the ratio (CHR/DNA; thick black line) within 1.2 kb of flanking chromatin relative to ATG sites of all 12,750 genes. An ∼170-bp nucleosome-depleted (“free”) region (NDR) is centered near the AT-rich regions of Dictyostelium TSS. Positioned nucleosomes upstream (+) and downstream (−) to the NDR are indicated by arrows. The protein cds region is shaded.

    Journal: Genome Research

    Article Title: Regulation of nucleosome positioning by a CHD Type III chromatin remodeler and its relationship to developmental gene expression in Dictyostelium

    doi: 10.1101/gr.216309.116

    Figure Lengend Snippet: Genome-wide nucleosome positioning in Dictyostelium . ( A ) Normalized read midpoint frequency distributions of MNase-protected fragments (nucleosome dyads) of all 12,750 genes in growth-stage WT cells were aligned relative to their ATG codons. Peaks (arrows) correspond to dyad midpoints for globally phased nucleosomes in the 5′ region of intragenic DNA, and distances between mapped read peaks correspond to ∼170 bp NRL. The protein coding DNA sequence (cds) region is shaded. ( B ) Normalized read midpoint frequency distributions of all genes in growth-stage WT cells were aligned relative to their translational termination sites (stop codons). Peaks (arrows) in the mean normalized frequency distribution correspond to globally phased nucleosomes in the 3′ region of intragenic DNA. The protein cds region is shaded. ( C ) Normalized dyad read midpoint frequency distributions for WT chromatin (CHR; dotted line) (see A ) were adjusted for sequence mappability by dividing with equivalent control data from MNase-digested naked (protein free) WT DNA (DNA; red line) and replotted as the ratio (CHR/DNA; thick black line) within 1.2 kb of flanking chromatin relative to ATG sites of all 12,750 genes. An ∼170-bp nucleosome-depleted (“free”) region (NDR) is centered near the AT-rich regions of Dictyostelium TSS. Positioned nucleosomes upstream (+) and downstream (−) to the NDR are indicated by arrows. The protein cds region is shaded.

    Article Snippet: Chromatin was digested with micrococcal nuclease (MNase) to create nuclease-resistant DNA ladders with a fragment spectrum of < 1 kb and was analyzed by Illumina paired-end DNA sequencing.

    Techniques: Genome Wide, Sequencing

    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

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

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.1000639

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

    Article Snippet: NETs from 10 wells were digested with 5 U/ml MNase (Fermentas), a non-processive nuclease that cuts DNA at linker sites.

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

    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

    Chromatin-remodeling (nucleosome insertion and rearrangement) on Nanog gene promoter. ( A ) ESCs were treated with RA for 3 days, then treated with 5, 10 and 30 U of MNase for 6 min at 37°C. Extracted chromatin DNA was separated on 1.5% agarose gels followed by Southern blot hybridization with 32 P-labeled 500-bp probes specific to three regions on the Nanog promoter. Positions of probes are depicted on the map. ( B ) Restriction enzyme accessibility of Nanog promoter region in ESCs. Asterisk marks the diagnostic band indicative of each sensitive site. The results are summarized and shown on the map above these blots. Restriction sites are labeled under the map. ( C ) N1, N2, N3 and N4 PCR fragments, amplified from mononucleosomal DNA using the primers a/b (N1), c/d (N2), e/f (N3) and g/h (N4). Primer sequences were listed in Supplementary Table S1 . Genomic DNAs isolated from 1 × 10 6 cells were used for N1 amplification as control (upper panel). ESCs were transfected with siRNA specific for Brm or siRNA control. Changes in mononucleosome formation on the Nanog gene promoter were monitored (bottom panel). ( D ) Upper: nucleosome occupancy on the Nanog promoter in ESCs with or without RA treatment. The gray-highlighted region (q8–12) represents the location of the N2 nucleosome formation. Lower: analysis of nucleosome occupancy at the q8–12 regions of Nanog in control and Brm-knockdown ES cell by the MNase resistance assay. Data points represent average qPCR signals from two independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Coordinated repressive chromatin-remodeling of Oct4 and Nanog genes in RA-induced differentiation of embryonic stem cells involves RIP140

    doi: 10.1093/nar/gku092

    Figure Lengend Snippet: Chromatin-remodeling (nucleosome insertion and rearrangement) on Nanog gene promoter. ( A ) ESCs were treated with RA for 3 days, then treated with 5, 10 and 30 U of MNase for 6 min at 37°C. Extracted chromatin DNA was separated on 1.5% agarose gels followed by Southern blot hybridization with 32 P-labeled 500-bp probes specific to three regions on the Nanog promoter. Positions of probes are depicted on the map. ( B ) Restriction enzyme accessibility of Nanog promoter region in ESCs. Asterisk marks the diagnostic band indicative of each sensitive site. The results are summarized and shown on the map above these blots. Restriction sites are labeled under the map. ( C ) N1, N2, N3 and N4 PCR fragments, amplified from mononucleosomal DNA using the primers a/b (N1), c/d (N2), e/f (N3) and g/h (N4). Primer sequences were listed in Supplementary Table S1 . Genomic DNAs isolated from 1 × 10 6 cells were used for N1 amplification as control (upper panel). ESCs were transfected with siRNA specific for Brm or siRNA control. Changes in mononucleosome formation on the Nanog gene promoter were monitored (bottom panel). ( D ) Upper: nucleosome occupancy on the Nanog promoter in ESCs with or without RA treatment. The gray-highlighted region (q8–12) represents the location of the N2 nucleosome formation. Lower: analysis of nucleosome occupancy at the q8–12 regions of Nanog in control and Brm-knockdown ES cell by the MNase resistance assay. Data points represent average qPCR signals from two independent experiments.

    Article Snippet: Nuclei isolated from ESCs were digested with 5 and 30 U of MNase (Worthington, Lakewood, NJ, USA, www.worthington-biochem.com ) at 37°C for 5 min, followed by proteinase K treatment at 37°C overnight.

    Techniques: Southern Blot, Hybridization, Labeling, Diagnostic Assay, Polymerase Chain Reaction, Amplification, Isolation, Transfection, Real-time Polymerase Chain Reaction

    Differential chromatin structure and transcription factor binding between consensus and Alu RBPJ binding sites. ( A ) Heat map of clustered reads densities for the indicated genome-wide determination or DNA sequence feature centered around RBPJ peak summits for all 28 220 RBPJ binding sites. ( B ) The same analysis as in A for the 4921 RBPJ peak summits that intersected with an Alu element within 200 bp. ( C ) Quantification of DNA ends densities for MNAse digested input, RBPJ immunoprecipitated, and DNase hypersensitive DNA for cluster 1 (without Alu) and cluster 5 (with Alu) regions.

    Journal: Nucleic Acids Research

    Article Title: RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF

    doi: 10.1093/nar/gky562

    Figure Lengend Snippet: Differential chromatin structure and transcription factor binding between consensus and Alu RBPJ binding sites. ( A ) Heat map of clustered reads densities for the indicated genome-wide determination or DNA sequence feature centered around RBPJ peak summits for all 28 220 RBPJ binding sites. ( B ) The same analysis as in A for the 4921 RBPJ peak summits that intersected with an Alu element within 200 bp. ( C ) Quantification of DNA ends densities for MNAse digested input, RBPJ immunoprecipitated, and DNase hypersensitive DNA for cluster 1 (without Alu) and cluster 5 (with Alu) regions.

    Article Snippet: Cells were fixed with 0.7% formaldehyde and DNA was digested by 10 ul MNAse (New England Biolabs) to an average length of 120 bp.

    Techniques: Binding Assay, Genome Wide, Sequencing, Immunoprecipitation

    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

    Fun30 is required for normal CEN -flanking nucleosome positioning and/or CEN core particle structure. A) Genome browser trace of Fun30 ChIP enrichment and nucleosome dyad frequency centred on and surrounding yeast CEN1 . The upper trace shows Log 2 Fun30 ChIP-seq enrichment values binned at 10 bp intervals and smoothed with a 3 bin moving average. Wildtype (WT) and Δfun30 chromatin was digested with MNase and nuclease-protected DNA species sequenced using paired-end mode Illumina technology. Nucleosome sequencing data (nuc) traces were plotted as mirror images in the lower panel. The graph shows a map of the centre point positions of paired sequence reads with end-to-end distances of 150 bp+/−20% wild-type and Δfun30 mutant chromatin samples surrounding CEN1 . The frequency distributions, which effectively map chromatin particle dyads, were binned at 10 bp intervals, and smoothed by applying a 3 bin moving average. Peaks in the dyad distributions correspond to translationally-positioned nucleosomes in the original genome. The CEN core particle is also mapped using this method and can be visualised as a small peak centred on the CEN region marked with a grey box. Pink bars show the positions of ORFs (B–D) Genome browser plots of Fun30 ChIP-seq and nucleosome sequence distributions as described above for CEN10, 11 and 12 respectively. Fun30-dependent changes in the height of a nucleosome dyad or CEN core particle peak are marked with a red asterix. Fun30-dependent changes in the position of a CEN -flanking nucleosome dyad peak are marked with red arrows. Genome browser plots for all yeast CENs are shown in Figure S6 .

    Journal: PLoS Genetics

    Article Title: SWI/SNF-Like Chromatin Remodeling Factor Fun30 Supports Point Centromere Function in S. cerevisiae

    doi: 10.1371/journal.pgen.1002974

    Figure Lengend Snippet: Fun30 is required for normal CEN -flanking nucleosome positioning and/or CEN core particle structure. A) Genome browser trace of Fun30 ChIP enrichment and nucleosome dyad frequency centred on and surrounding yeast CEN1 . The upper trace shows Log 2 Fun30 ChIP-seq enrichment values binned at 10 bp intervals and smoothed with a 3 bin moving average. Wildtype (WT) and Δfun30 chromatin was digested with MNase and nuclease-protected DNA species sequenced using paired-end mode Illumina technology. Nucleosome sequencing data (nuc) traces were plotted as mirror images in the lower panel. The graph shows a map of the centre point positions of paired sequence reads with end-to-end distances of 150 bp+/−20% wild-type and Δfun30 mutant chromatin samples surrounding CEN1 . The frequency distributions, which effectively map chromatin particle dyads, were binned at 10 bp intervals, and smoothed by applying a 3 bin moving average. Peaks in the dyad distributions correspond to translationally-positioned nucleosomes in the original genome. The CEN core particle is also mapped using this method and can be visualised as a small peak centred on the CEN region marked with a grey box. Pink bars show the positions of ORFs (B–D) Genome browser plots of Fun30 ChIP-seq and nucleosome sequence distributions as described above for CEN10, 11 and 12 respectively. Fun30-dependent changes in the height of a nucleosome dyad or CEN core particle peak are marked with a red asterix. Fun30-dependent changes in the position of a CEN -flanking nucleosome dyad peak are marked with red arrows. Genome browser plots for all yeast CENs are shown in Figure S6 .

    Article Snippet: MNase digested chromatin samples processed for paired-end mode Illumina DNA sequencing.

    Techniques: Chromatin Immunoprecipitation, Sequencing, Mutagenesis