Structured Review

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 .
Micrococcal Nuclease Mnase, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Reassembly of Nucleosomes at the MLH1 Promoter Initiates Resilencing Following Decitabine Exposure"

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

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1003636

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 .
Figure Legend 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 .

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

Related Articles

Centrifugation:

Article Title: Identification of an Intermediate in Hepatitis B Virus Covalently Closed Circular (CCC) DNA Formation and Sensitive and Selective CCC DNA Detection
Article Snippet: .. After removal of the nuclear pellet by centrifugation, the supernatant (cytoplasmic lysate) was incubated with micrococcal nuclease (MNase) (Roche) (final concentration, 150 units/ml) and CaCl2 (final concentration, 5 mM) at 37°C for 90 min to degrade the nucleic acids outside nucleocapsids. .. The MNase was then inactivated by addition of EDTA to reach a concentration of 10 mM.

Isolation:

Article Title: Reassembly of Nucleosomes at the MLH1 Promoter Initiates Resilencing Following Decitabine Exposure
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). .. Chromatin was digested using 20 U MNase (Fermentas) for 4 min at 37°C to achieve maximal digestion to mononucleosomes.

Real-time Polymerase Chain Reaction:

Article Title: Reassembly of Nucleosomes at the MLH1 Promoter Initiates Resilencing Following Decitabine Exposure
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). .. Chromatin was digested using 20 U MNase (Fermentas) for 4 min at 37°C to achieve maximal digestion to mononucleosomes.

Concentration Assay:

Article Title: Identification of an Intermediate in Hepatitis B Virus Covalently Closed Circular (CCC) DNA Formation and Sensitive and Selective CCC DNA Detection
Article Snippet: .. After removal of the nuclear pellet by centrifugation, the supernatant (cytoplasmic lysate) was incubated with micrococcal nuclease (MNase) (Roche) (final concentration, 150 units/ml) and CaCl2 (final concentration, 5 mM) at 37°C for 90 min to degrade the nucleic acids outside nucleocapsids. .. The MNase was then inactivated by addition of EDTA to reach a concentration of 10 mM.

Article Title: Aspartic Proteases and Major Cell Wall Components in Candida albicans Trigger the Release of Neutrophil Extracellular Traps
Article Snippet: .. A 400 μl aliquot of micrococcal nuclease (MNase) at a concentration of 1 U/ml (Roche) was then added and the cells were incubated at 37°C for an additional 20 min to digest any released DNA. .. The nuclease activity was stopped by the addition of 5 mM EDTA.

Article Title: Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients
Article Snippet: .. To isolate intracellular capsid-associated viral RNA, HepAD38 cells were lysed in NP-40 lysis buffer (50 mM Tris-Cl [pH 7.8], 1 mM EDTA, 1% NP-40), and cytoplasmic lysates were incubated with CaCl2 (final concentration, 5 mM) and micrococcal nuclease (MNase) (Roche) (final concentration, 15 U/ml) at 37°C for 1 h to remove nucleic acids outside nucleocapsids. .. The reaction was terminated by addition of EDTA (final concentration, 15 mM), and then proteinase K (0.5 mg/ml without SDS) was added to the mixture, followed by incubation at 37°C for 30 min to inactivate MNase.

Incubation:

Article Title: Identification of an Intermediate in Hepatitis B Virus Covalently Closed Circular (CCC) DNA Formation and Sensitive and Selective CCC DNA Detection
Article Snippet: .. After removal of the nuclear pellet by centrifugation, the supernatant (cytoplasmic lysate) was incubated with micrococcal nuclease (MNase) (Roche) (final concentration, 150 units/ml) and CaCl2 (final concentration, 5 mM) at 37°C for 90 min to degrade the nucleic acids outside nucleocapsids. .. The MNase was then inactivated by addition of EDTA to reach a concentration of 10 mM.

Article Title: Wobble modification differences and subcellular localization of tRNAs in Leishmania tarentolae: implication for tRNA sorting mechanism
Article Snippet: .. After incubation at 27°C for 5 min, 100 U of micrococcal nuclease (MNase) (Roche) and 5 mM CaCl2 were added to digest the RNAs that were not imported into the mitochondria. ..

Article Title: Aspartic Proteases and Major Cell Wall Components in Candida albicans Trigger the Release of Neutrophil Extracellular Traps
Article Snippet: .. A 400 μl aliquot of micrococcal nuclease (MNase) at a concentration of 1 U/ml (Roche) was then added and the cells were incubated at 37°C for an additional 20 min to digest any released DNA. .. The nuclease activity was stopped by the addition of 5 mM EDTA.

Article Title: Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients
Article Snippet: .. To isolate intracellular capsid-associated viral RNA, HepAD38 cells were lysed in NP-40 lysis buffer (50 mM Tris-Cl [pH 7.8], 1 mM EDTA, 1% NP-40), and cytoplasmic lysates were incubated with CaCl2 (final concentration, 5 mM) and micrococcal nuclease (MNase) (Roche) (final concentration, 15 U/ml) at 37°C for 1 h to remove nucleic acids outside nucleocapsids. .. The reaction was terminated by addition of EDTA (final concentration, 15 mM), and then proteinase K (0.5 mg/ml without SDS) was added to the mixture, followed by incubation at 37°C for 30 min to inactivate MNase.

Article Title: Hepatitis B Virus Covalently Closed Circular DNA Formation in Immortalized Mouse Hepatocytes Associated with Nucleocapsid Destabilization
Article Snippet: .. The resulting cytoplasmic lysate was incubated with micrococcal nuclease (MNase) (Roche) (150 units/ml) and CaCl2 (5 mM) at 37°C for 90 min to degrade the nucleic acids outside NCs. ..

Protease Inhibitor:

Article Title: Reassembly of Nucleosomes at the MLH1 Promoter Initiates Resilencing Following Decitabine Exposure
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). .. Chromatin was digested using 20 U MNase (Fermentas) for 4 min at 37°C to achieve maximal digestion to mononucleosomes.

Lysis:

Article Title: Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients
Article Snippet: .. To isolate intracellular capsid-associated viral RNA, HepAD38 cells were lysed in NP-40 lysis buffer (50 mM Tris-Cl [pH 7.8], 1 mM EDTA, 1% NP-40), and cytoplasmic lysates were incubated with CaCl2 (final concentration, 5 mM) and micrococcal nuclease (MNase) (Roche) (final concentration, 15 U/ml) at 37°C for 1 h to remove nucleic acids outside nucleocapsids. .. The reaction was terminated by addition of EDTA (final concentration, 15 mM), and then proteinase K (0.5 mg/ml without SDS) was added to the mixture, followed by incubation at 37°C for 30 min to inactivate MNase.

Chromatin Immunoprecipitation:

Article Title: H2B ubiquitylation is part of chromatin architecture that marks exon-intron structure in budding yeast
Article Snippet: .. For all ChIP experiments analyzing individual genes, cell lysates were digested with 160 U of Micrococcal nuclease (MNase) per 100 ml of cells for 15 min (Nuclease S7, Roche Applied Science, Taiwan). .. The following antibodies were used with the equivalence of 10 OD units of cell lysate: FLAG, 20 μl (M2-F3165; Sigma-Aldrich, St. Louis, MO); HA, 5 μl (3F10; Roche Applied Science, Taiwan); H3, 4 μl (ab1791; Abcam, Cambridge, England); H3K4me3, 2 μl (ab8580; Abcam); H3K79me3, 10 μl (ab 2621, Abcam); and H3K36me3, 4 μl (ab9050, Abcam).

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  • mnase  (Roche)
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    Roche mnase
    SAMOSA captures bulk and single-molecule evidence of transcription factor-DNA interaction simultaneously via two orthogonal molecular signals. A.-F.) SAMOSA <t>MNase-cut</t> signal averaged over predicted CTCF, NRF1, REST, PU.1, c-MYC, and GATA1 binding motifs in the <t>K562</t> epigenome. All binding sites were predicted from ENCODE ChIP-seq data. G-L.) m 6 dA signal for the same transcription factors, averaged over molecules containing predicted binding sites and at least 250 bases flanking DNA on either side of the predicted motif. Methylation patterns at predicted sites were compared against average profiles taken from randomly drawn molecules from GC%- and repeat-content-matched regions of the genome (calculated for each ENCODE ChIP-seq peak set). M.) Results of clustering motif-containing molecules using the Leiden community detection algorithm. Clusters were manually annotated as containing molecules that were: ‘methylation resistant’ (MR), nucleosome occupied (NO1-8), stochastically accessible (SA1-2), accessible (A), or hyper-accessible (HA). N.) Heatmap representation of single-molecule accessibility profiles for clusters NO7, NO8, and A (500 randomly sampled molecules per cluster). O.) Our data may be explained by the Widom ‘site exposure’ model in vivo . Transcription factor binding motifs are stochastically exposed as nucleosomes toggle between multiple ‘registers’ as seen in Figure 3M (states NO and SA). Transcription factor binding perhaps enforces a favorable nucleosome register (state A), which can then seed hyper-accessible states / further TF-DNA interactions (state HA).
    Mnase, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Roche mnase digested dna
    Humanized yeast have trouble adapting to new conditions (A) Violin plots showing that humanized yeast cells are larger and have dysregulated cell size based on phase-contrast microscopy measurements. (B) Cell-cycle ( CLB1 ) and cell-size ( WHI4 ) regulating genes each have highly occupied −1 and −2 nucleosomes. (C) Humanized yeast have a prolonged S-phase and/or arrest in G1. Cell-cycle was analyzed by sytox green straining of <t>DNA</t> content and measured by flow cytometry. Each plot shows 10,000 cells. (D) Humanized yeast have delayed remodeling at the GAL1 promoter. Time-course was analyzed by galactose induction of eGFP using flow cytometry. (E) <t>MNase-seq</t> map of PHO5 promoter, and time-course nucleosome scanning assay using WT, hH3.1-core, and hH3.3-core nucleosome yeasts upon phosphate starvation at different time points. Data points show qPCR amplicon midpoints, and mean ± SD of 2 biological replicates.
    Mnase Digested Dna, supplied by Roche, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SAMOSA captures bulk and single-molecule evidence of transcription factor-DNA interaction simultaneously via two orthogonal molecular signals. A.-F.) SAMOSA MNase-cut signal averaged over predicted CTCF, NRF1, REST, PU.1, c-MYC, and GATA1 binding motifs in the K562 epigenome. All binding sites were predicted from ENCODE ChIP-seq data. G-L.) m 6 dA signal for the same transcription factors, averaged over molecules containing predicted binding sites and at least 250 bases flanking DNA on either side of the predicted motif. Methylation patterns at predicted sites were compared against average profiles taken from randomly drawn molecules from GC%- and repeat-content-matched regions of the genome (calculated for each ENCODE ChIP-seq peak set). M.) Results of clustering motif-containing molecules using the Leiden community detection algorithm. Clusters were manually annotated as containing molecules that were: ‘methylation resistant’ (MR), nucleosome occupied (NO1-8), stochastically accessible (SA1-2), accessible (A), or hyper-accessible (HA). N.) Heatmap representation of single-molecule accessibility profiles for clusters NO7, NO8, and A (500 randomly sampled molecules per cluster). O.) Our data may be explained by the Widom ‘site exposure’ model in vivo . Transcription factor binding motifs are stochastically exposed as nucleosomes toggle between multiple ‘registers’ as seen in Figure 3M (states NO and SA). Transcription factor binding perhaps enforces a favorable nucleosome register (state A), which can then seed hyper-accessible states / further TF-DNA interactions (state HA).

    Journal: bioRxiv

    Article Title: Massively multiplex single-molecule oligonucleosome footprinting

    doi: 10.1101/2020.05.20.105379

    Figure Lengend Snippet: SAMOSA captures bulk and single-molecule evidence of transcription factor-DNA interaction simultaneously via two orthogonal molecular signals. A.-F.) SAMOSA MNase-cut signal averaged over predicted CTCF, NRF1, REST, PU.1, c-MYC, and GATA1 binding motifs in the K562 epigenome. All binding sites were predicted from ENCODE ChIP-seq data. G-L.) m 6 dA signal for the same transcription factors, averaged over molecules containing predicted binding sites and at least 250 bases flanking DNA on either side of the predicted motif. Methylation patterns at predicted sites were compared against average profiles taken from randomly drawn molecules from GC%- and repeat-content-matched regions of the genome (calculated for each ENCODE ChIP-seq peak set). M.) Results of clustering motif-containing molecules using the Leiden community detection algorithm. Clusters were manually annotated as containing molecules that were: ‘methylation resistant’ (MR), nucleosome occupied (NO1-8), stochastically accessible (SA1-2), accessible (A), or hyper-accessible (HA). N.) Heatmap representation of single-molecule accessibility profiles for clusters NO7, NO8, and A (500 randomly sampled molecules per cluster). O.) Our data may be explained by the Widom ‘site exposure’ model in vivo . Transcription factor binding motifs are stochastically exposed as nucleosomes toggle between multiple ‘registers’ as seen in Figure 3M (states NO and SA). Transcription factor binding perhaps enforces a favorable nucleosome register (state A), which can then seed hyper-accessible states / further TF-DNA interactions (state HA).

    Article Snippet: Isolation of nuclei, MNase digest, and overnight dialysis 100E6 K562 cells were collected by centrifugation (300x g , 5 min), washed in ice cold 1X PBS, and resuspended in 1 mL Nuclear Isolation Buffer (20mM HEPES, 10mM KCl, 1mM MgCl2, 0.1% Triton X-100, 20% Glycerol, and 1X Protease Inhibitor (Roche)) per 5-10 e6 cells by gently pipetting 5x with a wide-bore tip to release nuclei.

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Methylation, In Vivo

    In vivo SAMOSA captures oligonucleosome structure by combining MNase digestion of chromatin with adenine methylation footprinting. A.) An overview of the in vivo SAMOSA protocol: oligonucleosomes are gently solubilized from nuclei using micrococcal nuclease and fusogenic lipid treatment. Resulting oligonucleosomes are footprinted using the EcoGII enzyme and sequencing on the PacBio platform. Each sequencing molecules captures two orthogonal biological signals: MNase cuts that capture ‘barrier’ protein-DNA interactions, and m 6 dA methylation protein-DNA footprints. B.) Fragment length distributions for in vivo SAMOSA data reveal expected oligonucleosomal laddering (bin size = 5 bp). C.) Averaged modification probabilities from SAMOSA experiments demonstrate the ability to mark nucleosome-DNA interactions directly via methylation. Modification patterns seen in the chromatin sample are not seen in unmethylated oligonucleosomal DNA or fully methylated K562 oligonucleosomal DNA.

    Journal: bioRxiv

    Article Title: Massively multiplex single-molecule oligonucleosome footprinting

    doi: 10.1101/2020.05.20.105379

    Figure Lengend Snippet: In vivo SAMOSA captures oligonucleosome structure by combining MNase digestion of chromatin with adenine methylation footprinting. A.) An overview of the in vivo SAMOSA protocol: oligonucleosomes are gently solubilized from nuclei using micrococcal nuclease and fusogenic lipid treatment. Resulting oligonucleosomes are footprinted using the EcoGII enzyme and sequencing on the PacBio platform. Each sequencing molecules captures two orthogonal biological signals: MNase cuts that capture ‘barrier’ protein-DNA interactions, and m 6 dA methylation protein-DNA footprints. B.) Fragment length distributions for in vivo SAMOSA data reveal expected oligonucleosomal laddering (bin size = 5 bp). C.) Averaged modification probabilities from SAMOSA experiments demonstrate the ability to mark nucleosome-DNA interactions directly via methylation. Modification patterns seen in the chromatin sample are not seen in unmethylated oligonucleosomal DNA or fully methylated K562 oligonucleosomal DNA.

    Article Snippet: Isolation of nuclei, MNase digest, and overnight dialysis 100E6 K562 cells were collected by centrifugation (300x g , 5 min), washed in ice cold 1X PBS, and resuspended in 1 mL Nuclear Isolation Buffer (20mM HEPES, 10mM KCl, 1mM MgCl2, 0.1% Triton X-100, 20% Glycerol, and 1X Protease Inhibitor (Roche)) per 5-10 e6 cells by gently pipetting 5x with a wide-bore tip to release nuclei.

    Techniques: In Vivo, Methylation, Footprinting, Sequencing, Modification

    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

    Nuclease and PK digestion of fractionated and unfractionated NCs. (A) Sucrose gradient fractions 6, 9, and 12 prepared from lysates without prior MNase treatment () were digested with MNase or DNase I as described in Materials and Methods. The reaction

    Journal: Journal of Virology

    Article Title: Maturation-Associated Destabilization of Hepatitis B Virus Nucleocapsid

    doi: 10.1128/JVI.01912-13

    Figure Lengend Snippet: Nuclease and PK digestion of fractionated and unfractionated NCs. (A) Sucrose gradient fractions 6, 9, and 12 prepared from lysates without prior MNase treatment () were digested with MNase or DNase I as described in Materials and Methods. The reaction

    Article Snippet: NCs isolated by sucrose gradient centrifugation (10 μl of the indicated sucrose fraction) or cytoplasmic lysate (10 μl) were treated with 1 mg/ml of PK (Invitrogen) (unless noted otherwise) in EPR buffer in a total volume of 20 μl at 37°C for 1 h. Similarly, for nuclease treatment, MNase (0.25 unit/μl) or DNase I (Roche) (2 mg/ml) was added, and the reaction mixture was incubated at 37°C for 1 h. For MNase digestion, the MgCl2 in the EPR buffer was replaced with CaCl2 (5 mM).

    Techniques:

    Humanized yeast have trouble adapting to new conditions (A) Violin plots showing that humanized yeast cells are larger and have dysregulated cell size based on phase-contrast microscopy measurements. (B) Cell-cycle ( CLB1 ) and cell-size ( WHI4 ) regulating genes each have highly occupied −1 and −2 nucleosomes. (C) Humanized yeast have a prolonged S-phase and/or arrest in G1. Cell-cycle was analyzed by sytox green straining of DNA content and measured by flow cytometry. Each plot shows 10,000 cells. (D) Humanized yeast have delayed remodeling at the GAL1 promoter. Time-course was analyzed by galactose induction of eGFP using flow cytometry. (E) MNase-seq map of PHO5 promoter, and time-course nucleosome scanning assay using WT, hH3.1-core, and hH3.3-core nucleosome yeasts upon phosphate starvation at different time points. Data points show qPCR amplicon midpoints, and mean ± SD of 2 biological replicates.

    Journal: Cell

    Article Title: Resetting the yeast epigenome with human nucleosomes

    doi: 10.1016/j.cell.2017.10.043

    Figure Lengend Snippet: Humanized yeast have trouble adapting to new conditions (A) Violin plots showing that humanized yeast cells are larger and have dysregulated cell size based on phase-contrast microscopy measurements. (B) Cell-cycle ( CLB1 ) and cell-size ( WHI4 ) regulating genes each have highly occupied −1 and −2 nucleosomes. (C) Humanized yeast have a prolonged S-phase and/or arrest in G1. Cell-cycle was analyzed by sytox green straining of DNA content and measured by flow cytometry. Each plot shows 10,000 cells. (D) Humanized yeast have delayed remodeling at the GAL1 promoter. Time-course was analyzed by galactose induction of eGFP using flow cytometry. (E) MNase-seq map of PHO5 promoter, and time-course nucleosome scanning assay using WT, hH3.1-core, and hH3.3-core nucleosome yeasts upon phosphate starvation at different time points. Data points show qPCR amplicon midpoints, and mean ± SD of 2 biological replicates.

    Article Snippet: Processed MNase digested DNA was analyzed by qPCR using primers listed in in a Roche LightCycler 1536 real-time PCR machine using LightCycler 1536 DNA Green Master (Roche) in technical triplicate.

    Techniques: Microscopy, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Amplification