mnase  (New England Biolabs)


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  • 99
    Name:
    Micrococcal Nuclease
    Description:
    Micrococcal Nuclease 320 000 gel units
    Catalog Number:
    M0247S
    Price:
    74
    Size:
    320 000 gel units
    Category:
    Exonucleases
    Score:
    85
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    Structured Review

    New England Biolabs mnase
    Micrococcal Nuclease
    Micrococcal Nuclease 320 000 gel units
    https://www.bioz.com/result/mnase/product/New England Biolabs
    Average 99 stars, based on 49 article reviews
    Price from $9.99 to $1999.99
    mnase - by Bioz Stars, 2019-12
    99/100 stars

    Images

    1) Product Images from "An Improved Strategy to Recover Large Fragments of Functional Human Neutrophil Extracellular Traps"

    Article Title: An Improved Strategy to Recover Large Fragments of Functional Human Neutrophil Extracellular Traps

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2013.00166

    DNA nucleases induce NET digestion . (A) Migration profile of pure λDNA after digestion with 4 U/mL DNase, MNase, or Alu-I. (B) Alu-I, DNase, and MNase dose-effects on NET dsDNA obtained after A23187 stimulation of PMN. Incubation with the restriction enzymes lasted 20 min at 37°C. DNA migration took place in 0.8% agarose gel containing ethidium bromide.
    Figure Legend Snippet: DNA nucleases induce NET digestion . (A) Migration profile of pure λDNA after digestion with 4 U/mL DNase, MNase, or Alu-I. (B) Alu-I, DNase, and MNase dose-effects on NET dsDNA obtained after A23187 stimulation of PMN. Incubation with the restriction enzymes lasted 20 min at 37°C. DNA migration took place in 0.8% agarose gel containing ethidium bromide.

    Techniques Used: Migration, Incubation, Agarose Gel Electrophoresis

    2) Product Images from "Nucleosomes Are Stably Evicted from Enhancers but Not Promoters upon Induction of Certain Pro-Inflammatory Genes in Mouse Macrophages"

    Article Title: Nucleosomes Are Stably Evicted from Enhancers but Not Promoters upon Induction of Certain Pro-Inflammatory Genes in Mouse Macrophages

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0093971

    PolII and TBP binding in the fraction of IL12B and IL1A promoters in a population of induced BMDMs that is nucleosome-free. (A and B), ChIP experiments were performed as described in the legend of Figure 2F with antibodies that detect (A) PolII or (B) TBP in BMDMs before (dark blue bars), and upon 1.5 h (yellow) or 3 h (red) LPS induction. Cross-linked chromatin was either untreated (solid bars), or lightly digested with MNase (hatched bars) as described in the Materials and Methods . The data was normalized to a region in the KIT promoter and genomic locations are indicated. The experiment was performed twice and error bars indicating the SEM are shown.
    Figure Legend Snippet: PolII and TBP binding in the fraction of IL12B and IL1A promoters in a population of induced BMDMs that is nucleosome-free. (A and B), ChIP experiments were performed as described in the legend of Figure 2F with antibodies that detect (A) PolII or (B) TBP in BMDMs before (dark blue bars), and upon 1.5 h (yellow) or 3 h (red) LPS induction. Cross-linked chromatin was either untreated (solid bars), or lightly digested with MNase (hatched bars) as described in the Materials and Methods . The data was normalized to a region in the KIT promoter and genomic locations are indicated. The experiment was performed twice and error bars indicating the SEM are shown.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation

    3) Product Images from "Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure"

    Article Title: Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku150

    Nucleosome duplexes are decoupled into mononucleosomes. ( A ) EMSA of purified nucleosome duplexes containing H3(T118ph) HO and mp2-187 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S4 ). ( B ) Quantification of fraction of nucleosome duplexes (squares), nucleosome (circles) and free DNA (diamond) species for the gel in (A) versus time. Error bars are the standard deviation of three independent experiments. ( C ) EMSA of purified nucleosome duplexes and altosomes containing H3(T118ph) HO and mp2-247 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert in part to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S5 ). ( D ) Quantification of the fraction of nucleosome duplexes (squares), altosomes (triangles), nucleosome (circles) and free DNA (diamond) species for the gel in (C) versus time. Error bars are the standard deviation of three independent experiments.
    Figure Legend Snippet: Nucleosome duplexes are decoupled into mononucleosomes. ( A ) EMSA of purified nucleosome duplexes containing H3(T118ph) HO and mp2-187 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S4 ). ( B ) Quantification of fraction of nucleosome duplexes (squares), nucleosome (circles) and free DNA (diamond) species for the gel in (A) versus time. Error bars are the standard deviation of three independent experiments. ( C ) EMSA of purified nucleosome duplexes and altosomes containing H3(T118ph) HO and mp2-247 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert in part to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S5 ). ( D ) Quantification of the fraction of nucleosome duplexes (squares), altosomes (triangles), nucleosome (circles) and free DNA (diamond) species for the gel in (C) versus time. Error bars are the standard deviation of three independent experiments.

    Techniques Used: Purification, Standard Deviation

    4) Product Images from "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"

    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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky562

    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.
    Figure Legend 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.

    Techniques Used: Binding Assay, Genome Wide, Sequencing, Immunoprecipitation

    5) Product Images from "HITS-CLIP Analysis Uncovers a Link between the Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein and Host Pre-mRNA Metabolism"

    Article Title: HITS-CLIP Analysis Uncovers a Link between the Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein and Host Pre-mRNA Metabolism

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004652

    Isolation of cross-linked ORF57-RNA complexes for HITS-CLIP analysis. Top Nitrocellulose membrane from an ORF57 immunoprecipitation performed under HITS-CLIP conditions. Cross-linked RNA fragments were end-labeled with 32 P to be visualized by Phosphoimager. Cells were induced to undergo lytic reactivation, exposed to UV and/or treated with high or low concentrations of MNase as indicated. The samples lacking an ORF57 antibody (lane 1) were precipitated with pre-bleed antibodies from the same rabbit. Lanes 6 and 7 are a dark exposure of lanes 4 and 5. The dashed white box indicates the position of the ORF57 complex cut from the membrane for library preparation. Bottom Western blot of the same HITS-CLIP samples shown in the top panel. Affinity purified rabbit anti-ORF57 was used to detect ORF57. Positions of molecular weight markers are shown on the left. On the right, a single asterisk marks the position of a contaminating ~37 kDa protein; double and triple asterisks mark positions of putative ORF57 homodimers and homotrimers bound to the same RNA. The doublet is likely due to an ORF57 cleavage product [ 104 ].
    Figure Legend Snippet: Isolation of cross-linked ORF57-RNA complexes for HITS-CLIP analysis. Top Nitrocellulose membrane from an ORF57 immunoprecipitation performed under HITS-CLIP conditions. Cross-linked RNA fragments were end-labeled with 32 P to be visualized by Phosphoimager. Cells were induced to undergo lytic reactivation, exposed to UV and/or treated with high or low concentrations of MNase as indicated. The samples lacking an ORF57 antibody (lane 1) were precipitated with pre-bleed antibodies from the same rabbit. Lanes 6 and 7 are a dark exposure of lanes 4 and 5. The dashed white box indicates the position of the ORF57 complex cut from the membrane for library preparation. Bottom Western blot of the same HITS-CLIP samples shown in the top panel. Affinity purified rabbit anti-ORF57 was used to detect ORF57. Positions of molecular weight markers are shown on the left. On the right, a single asterisk marks the position of a contaminating ~37 kDa protein; double and triple asterisks mark positions of putative ORF57 homodimers and homotrimers bound to the same RNA. The doublet is likely due to an ORF57 cleavage product [ 104 ].

    Techniques Used: Isolation, Cross-linking Immunoprecipitation, Immunoprecipitation, Labeling, Western Blot, Affinity Purification, Molecular Weight

    6) Product Images from "CAL1 is the Drosophila CENP-A assembly factor"

    Article Title: CAL1 is the Drosophila CENP-A assembly factor

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201305036

    CAL1 is a CENP-A–specific nucleosome assembly factor. (A) CENP-A 101–225 –H4 was assembled on circular relaxed plasmid (pCR2.1-4 [CEN3 + CEN6]) using increasing amounts of CAL1 1–96 , CAL1 1–132 , or CAL1 1–160 in the presence of topoisomerase I. The extracted DNA samples were analyzed on agarose gel and stained with SYBR gold. Lane 1: supercoiled plasmid; lane 2: relaxed plasmid; lane 3: CENP-A 101–225 –H4, H2A–H2B but no CAL1; lanes 4–7: relaxed plasmid and CENP-A 101–225 –H4, H2A–H2B with CAL1 1–96 ; lanes 8–11: CENP-A 101–225 –H4, H2A–H2B with CAL1 1–132 ; lanes 12–15: CENP-A 101–225 –H4, H2A–H2B with CAL1 1–160 . (B) CENP-A–containing nucleosomes and histone H3 nucleosomes were assembled using histone proteins, CAL1 1–160 , or yeast Nap1 and pGEM3Z-601 plasmid. Lane 1: supercoiled plasmid; lane 2: relaxed plasmid; lanes 3, 6, and 9: mock reaction without CAL1 1–160 or Nap1; lanes 4 and 5: H3–H4, H2A–H2B with CAL1 1–160 ; lanes 7 and 8: CENP-A 101–225 –H4, H2A-H2B with CAL1 1–160 ; lanes 10 and 11: H3–H4, H2A–H2B with Nap1; lanes 12 and 13: CENP-A 101–225 –H4, H2A–H2B with Nap1. (C) CAL1-assembled CENP-A nucleosomes are negatively supercoiled. CENP-A nucleosomes (lane CENP-A) were assembled by CAL1 1–160 in the presence of topoisomerase I and H2A–H2B dimers, whereas histone H3 nucleosomes (lane H3) were assembled by Nap1. Controls are: supercoiled plasmid (superc.), relaxed plasmid (relaxed), and relaxed plasmid without histones or Nap1, but treated and processed as in the H3 and CENP-A nucleosome assembly reactions (mock). The samples were analyzed on agarose gels with or without 1 µg/ml chloroquine. Boxes highlight the decrease in migration that occurs in the presence of chloroquine in both H3 and CENP-A nucleosomes. (A–C) S, supercoiled; R, relaxed plasmid. (D) Diagram depicting the expected migration patterns for negatively and positively supercoiled DNA separated by 2D gel electrophoresis ( Tachiwana et al., 2011 ). Left gel: H3 nucleosomes assembled with Nap1; right gel: CENP-A nucleosomes assembled with CAL1 1–160 . (E) Mononucleosomes were assembled on 147-bp Widom DNA with Nap1 or CAL1 1–160 and digested with MNase for 30 or 120 s. Native PAGE of samples shown. Lanes 1–3: DNA only; lanes 4 and 5: H3 nucleosomes assembled by Nap1; lanes 6 and 7: CENP-A nucleosomes assembled by Nap1; lanes 8 and 9: CENP-A nucleosomes assembled by CAL1 1–160 .
    Figure Legend Snippet: CAL1 is a CENP-A–specific nucleosome assembly factor. (A) CENP-A 101–225 –H4 was assembled on circular relaxed plasmid (pCR2.1-4 [CEN3 + CEN6]) using increasing amounts of CAL1 1–96 , CAL1 1–132 , or CAL1 1–160 in the presence of topoisomerase I. The extracted DNA samples were analyzed on agarose gel and stained with SYBR gold. Lane 1: supercoiled plasmid; lane 2: relaxed plasmid; lane 3: CENP-A 101–225 –H4, H2A–H2B but no CAL1; lanes 4–7: relaxed plasmid and CENP-A 101–225 –H4, H2A–H2B with CAL1 1–96 ; lanes 8–11: CENP-A 101–225 –H4, H2A–H2B with CAL1 1–132 ; lanes 12–15: CENP-A 101–225 –H4, H2A–H2B with CAL1 1–160 . (B) CENP-A–containing nucleosomes and histone H3 nucleosomes were assembled using histone proteins, CAL1 1–160 , or yeast Nap1 and pGEM3Z-601 plasmid. Lane 1: supercoiled plasmid; lane 2: relaxed plasmid; lanes 3, 6, and 9: mock reaction without CAL1 1–160 or Nap1; lanes 4 and 5: H3–H4, H2A–H2B with CAL1 1–160 ; lanes 7 and 8: CENP-A 101–225 –H4, H2A-H2B with CAL1 1–160 ; lanes 10 and 11: H3–H4, H2A–H2B with Nap1; lanes 12 and 13: CENP-A 101–225 –H4, H2A–H2B with Nap1. (C) CAL1-assembled CENP-A nucleosomes are negatively supercoiled. CENP-A nucleosomes (lane CENP-A) were assembled by CAL1 1–160 in the presence of topoisomerase I and H2A–H2B dimers, whereas histone H3 nucleosomes (lane H3) were assembled by Nap1. Controls are: supercoiled plasmid (superc.), relaxed plasmid (relaxed), and relaxed plasmid without histones or Nap1, but treated and processed as in the H3 and CENP-A nucleosome assembly reactions (mock). The samples were analyzed on agarose gels with or without 1 µg/ml chloroquine. Boxes highlight the decrease in migration that occurs in the presence of chloroquine in both H3 and CENP-A nucleosomes. (A–C) S, supercoiled; R, relaxed plasmid. (D) Diagram depicting the expected migration patterns for negatively and positively supercoiled DNA separated by 2D gel electrophoresis ( Tachiwana et al., 2011 ). Left gel: H3 nucleosomes assembled with Nap1; right gel: CENP-A nucleosomes assembled with CAL1 1–160 . (E) Mononucleosomes were assembled on 147-bp Widom DNA with Nap1 or CAL1 1–160 and digested with MNase for 30 or 120 s. Native PAGE of samples shown. Lanes 1–3: DNA only; lanes 4 and 5: H3 nucleosomes assembled by Nap1; lanes 6 and 7: CENP-A nucleosomes assembled by Nap1; lanes 8 and 9: CENP-A nucleosomes assembled by CAL1 1–160 .

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Staining, Migration, Two-Dimensional Gel Electrophoresis, Electrophoresis, Clear Native PAGE

    7) Product Images from "Organization of DNA in a bacterial nucleoid"

    Article Title: Organization of DNA in a bacterial nucleoid

    Journal: BMC Microbiology

    doi: 10.1186/s12866-016-0637-3

    A possible model of low-level nucleoid organization. a A fragment of EM image of E. coli nucleoid (adapted from [ 21 ]). b Genomic DNA (gray) forms loops with A-tract clusters (cyan) located at apexes and MNase resistant fragments (black) occupying loop ‘stems’. c A blow-up of a DNA loop, containing several A-tracts (cyan) and two MNase resistant fragments (black)
    Figure Legend Snippet: A possible model of low-level nucleoid organization. a A fragment of EM image of E. coli nucleoid (adapted from [ 21 ]). b Genomic DNA (gray) forms loops with A-tract clusters (cyan) located at apexes and MNase resistant fragments (black) occupying loop ‘stems’. c A blow-up of a DNA loop, containing several A-tracts (cyan) and two MNase resistant fragments (black)

    Techniques Used: Electron Microscopy

    Analysis of the DNA fragments from in vivo MNase digestion of nucleoid in wild type E. coli ( a ) and in vitro MNase digestion of purified genomic DNA ( b ) in agarose gel. a Wild type cells with the empty vector (lanes 2, 3) and MNase-expressing vector (lanes 4-8) were supplemented with arabinose (lane 6), CaCl 2 (lane 5) or both (lanes 3, 7, 8). Digestion reactions were stopped 1 minute (lane 7) or 5 minutes (lanes 3, 5, 8) after CaCl 2 was added. Lanes 1 and 9 show DNA molecular weight marker. b Lane 1, purified wild type genomic DNA; lanes 2-6, a time course of in vitro MNase digestion of the wild type genomic DNA; lane 7, DNA molecular marker
    Figure Legend Snippet: Analysis of the DNA fragments from in vivo MNase digestion of nucleoid in wild type E. coli ( a ) and in vitro MNase digestion of purified genomic DNA ( b ) in agarose gel. a Wild type cells with the empty vector (lanes 2, 3) and MNase-expressing vector (lanes 4-8) were supplemented with arabinose (lane 6), CaCl 2 (lane 5) or both (lanes 3, 7, 8). Digestion reactions were stopped 1 minute (lane 7) or 5 minutes (lanes 3, 5, 8) after CaCl 2 was added. Lanes 1 and 9 show DNA molecular weight marker. b Lane 1, purified wild type genomic DNA; lanes 2-6, a time course of in vitro MNase digestion of the wild type genomic DNA; lane 7, DNA molecular marker

    Techniques Used: In Vivo, In Vitro, Purification, Agarose Gel Electrophoresis, Plasmid Preparation, Expressing, Molecular Weight, Marker

    Genome-wide distribution of sequenced tags. a Tag frequencies for the entire E. coli genome. Frequencies of tags mapped on the positive and negative strands are shown with red and blue bars respectively. a , c Schematic illustrations of tag cross-correlation ( b ) and auto-correlation analyses ( c ). MNase resistant fragments are shown with grey rectangles. Vertical red and blue arrows represent 5’-ends of the digestion fragments mapping to the DNA positive and negative strands respectively
    Figure Legend Snippet: Genome-wide distribution of sequenced tags. a Tag frequencies for the entire E. coli genome. Frequencies of tags mapped on the positive and negative strands are shown with red and blue bars respectively. a , c Schematic illustrations of tag cross-correlation ( b ) and auto-correlation analyses ( c ). MNase resistant fragments are shown with grey rectangles. Vertical red and blue arrows represent 5’-ends of the digestion fragments mapping to the DNA positive and negative strands respectively

    Techniques Used: Genome Wide

    8) Product Images from "Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum"

    Article Title: Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum

    Journal: EMBO Reports

    doi: 10.15252/embr.201846331

    Ectopically expressed histone H3p localizes to the nucleus during parasite asexual development and incorporates into nucleosomes Indirect immunofluorescence assays were performed to determine the localization of ectopically expressed PfH3p‐HA in ring (R), trophozoite (T), and schizont (S) stages of Plasmodium falciparum asexual growth. PfH3p‐HA was detected using anti‐HA antibodies (green) and endogenous histone H3 with anti‐histone H3 N‐terminal antibodies (red). DAPI (blue) was used to stain the nucleus. Scale bar = 5 μm. Nuclei isolated from wild‐type (WT) or PfH3p‐HA‐expressing (WT + PfH3p‐HA) schizont‐stage parasites were treated with 4 U/ml of micrococcal nuclease (MNase) for the indicated amounts of time, the DNA purified and migrated on a 2% agarose gel, and stained with ethidium bromide. Mononucleosomes purified after 10 min of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis and either stained with Coomassie Brilliant Blue (C.B.) or visualized by immunoblotting with anti‐HA (α‐HA) or anti‐C‐terminal histone H3 (α‐H3c) antibodies. Co‐immunoprecipitation (IP) experiments of purified mononucleosomes obtained from wild‐type (WT) or transfected (WT + PfH3p‐HA) schizont‐stage parasites were performed with either anti‐HA antibodies or mouse IgG. Immunoprecipitated products (right panel) were analyzed by immunoblotting using anti‐HA or anti‐histone H4 antibodies. Source data are available online for this figure.
    Figure Legend Snippet: Ectopically expressed histone H3p localizes to the nucleus during parasite asexual development and incorporates into nucleosomes Indirect immunofluorescence assays were performed to determine the localization of ectopically expressed PfH3p‐HA in ring (R), trophozoite (T), and schizont (S) stages of Plasmodium falciparum asexual growth. PfH3p‐HA was detected using anti‐HA antibodies (green) and endogenous histone H3 with anti‐histone H3 N‐terminal antibodies (red). DAPI (blue) was used to stain the nucleus. Scale bar = 5 μm. Nuclei isolated from wild‐type (WT) or PfH3p‐HA‐expressing (WT + PfH3p‐HA) schizont‐stage parasites were treated with 4 U/ml of micrococcal nuclease (MNase) for the indicated amounts of time, the DNA purified and migrated on a 2% agarose gel, and stained with ethidium bromide. Mononucleosomes purified after 10 min of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis and either stained with Coomassie Brilliant Blue (C.B.) or visualized by immunoblotting with anti‐HA (α‐HA) or anti‐C‐terminal histone H3 (α‐H3c) antibodies. Co‐immunoprecipitation (IP) experiments of purified mononucleosomes obtained from wild‐type (WT) or transfected (WT + PfH3p‐HA) schizont‐stage parasites were performed with either anti‐HA antibodies or mouse IgG. Immunoprecipitated products (right panel) were analyzed by immunoblotting using anti‐HA or anti‐histone H4 antibodies. Source data are available online for this figure.

    Techniques Used: Immunofluorescence, Staining, Isolation, Expressing, Purification, Agarose Gel Electrophoresis, Polyacrylamide Gel Electrophoresis, Immunoprecipitation, Transfection

    9) Product Images from "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"

    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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky562

    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.
    Figure Legend 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.

    Techniques Used: Binding Assay, Genome Wide, Sequencing, Immunoprecipitation

    10) Product Images from "Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics"

    Article Title: Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky526

    Dynamics of H2B K34ub nucleosomes. ( A ) Nucleosomes assembled on 147 bp 601 DNA were resolved in native PAGE under ionic and temperature conditions indicated on top. For the middle panel, although electrophoresis was performed at ∼26°C, the temperature at the glass surface was ∼35–37°C due to higher conductivity of the buffer. All gels were pre-electrophoresed in the relevant buffer. ( B ) Unmodified and H2B K34ub nucleosomes /hexasomes assembled on 177 bp 601 were digested with MNase at 26°C or 37°C and DNA was resolved in 6.5% PAGE and stained with SYBR Gold.
    Figure Legend Snippet: Dynamics of H2B K34ub nucleosomes. ( A ) Nucleosomes assembled on 147 bp 601 DNA were resolved in native PAGE under ionic and temperature conditions indicated on top. For the middle panel, although electrophoresis was performed at ∼26°C, the temperature at the glass surface was ∼35–37°C due to higher conductivity of the buffer. All gels were pre-electrophoresed in the relevant buffer. ( B ) Unmodified and H2B K34ub nucleosomes /hexasomes assembled on 177 bp 601 were digested with MNase at 26°C or 37°C and DNA was resolved in 6.5% PAGE and stained with SYBR Gold.

    Techniques Used: Clear Native PAGE, Electrophoresis, Polyacrylamide Gel Electrophoresis, Staining

    Related Articles

    Centrifugation:

    Article Title: N6-methyladenine is an epigenetic marker of mammalian early life stress
    Article Snippet: Chromatin shearing was obtained using 1000 gel units of Micrococcal Nuclease (MNase) (New England Biolabs; catalog #M0247S); samples were incubated with Proteinase K to ensure efficient shearing of DNA (between 300 bp–900 bp). .. Chromatin shearing was obtained using 1000 gel units of Micrococcal Nuclease (MNase) (New England Biolabs; catalog #M0247S); samples were incubated with Proteinase K to ensure efficient shearing of DNA (between 300 bp–900 bp).

    Article Title: TRF2 is recruited to the pre-initiation complex as a testis-specific subunit of TFIIA/ALF to promote haploid cell gene expression
    Article Snippet: The cell pellet was collected by centrifugation for 5 min at 4000 rpm, lysed in 5 volumes of lysis buffer (50 mM Tris pH8, 10 mM EDTA, 1% SDS, protease inhibitors) and sonicated for 10 min on a Covaris sonicator. .. The lysate was sonicated for 30 sec as described above, CaCl2 and MgCl2 were added to 5 mM final concentration each and digestion was performed with MNase (10 gel units/uL final) (M0247S, New England BioLabs) for 10 min of incubation.

    Article Title: Temporal Analysis of the Honey Bee Microbiome Reveals Four Novel Viruses and Seasonal Prevalence of Known Viruses, Nosema, and Crithidia
    Article Snippet: For DNA purification, Crithidia mellificae (∼106 trypanosomes/mL culture medium) were pelleted by centrifugation (800×g for 6 min) and washed with PBS prior to DNA extraction. .. Bees from Crithidia positive hives were homogenized by TissueLyser as above and DNA extracted using the DNeasy kit for the initial PCR screens, after suspension in either PBS or 1× Micrococcal Nuclease Buffer (NEB).

    Article Title: Transient Downregulation of Nanog and Oct4 Induced by DETA/NO Exposure in Mouse Embryonic Stem Cells Leads to Mesodermal/Endodermal Lineage Differentiation
    Article Snippet: Following centrifugation at 4°C for 5 min, supernatants were discarded and pellets were washed by gentle inversion with 10 mM Tris HCl buffer, pH 8.0 containing 15 mM NaCl, and 60 mM KCl and centrifuged for 5 min at 4°C. .. Pellets were then resuspended in washing buffer supplemented with 3 mM CaCl2 , protease inhibitors, 0.5 mM DTT, and 5–10 μ L of 1 : 200 micrococcal nuclease (New England BioLabs) and incubated for 20 min at 37°C with orbital shaking.

    Article Title: Androgen Receptor Serine 81 Phosphorylation Mediates Chromatin Binding and Transcriptional Activation
    Article Snippet: The supernatant was removed and saved, and the pellet was resuspended in 100 μl of TLB containing 50 m m of NaCl and gently shaken for 10 min in at 4 °C, followed by centrifugation at 11,000 × g for 5 min. .. The insoluble fraction (pellet left from cytoplasmic and nuclear protein extraction) was suspended in 40 μl of micrococcal nuclease digestion buffer (M0247, New England Biolabs), kept at room temperature, and treated with 600 units of micrococcal nuclease for 2 min.

    Amplification:

    Article Title: Nucleosome positioning in the regulatory region of SV40 chromatin correlates with the activation and repression of early and late transcription during infection
    Article Snippet: Following sonication the sample was purified using Zymo Research ChIP DNA Clean and Concentrator columns according to their protocol and eluted in 51 μl H2 O. Micrococcal nuclease digested chromatin was obtained by treating 200 μl of SV40 chromatin with micrococcal nuclease (New England Biolabs, M0247S) at 4 °C. .. Following sonication the sample was purified using Zymo Research ChIP DNA Clean and Concentrator columns according to their protocol and eluted in 51 μl H2 O. Micrococcal nuclease digested chromatin was obtained by treating 200 μl of SV40 chromatin with micrococcal nuclease (New England Biolabs, M0247S) at 4 °C.

    Polymerase Chain Reaction:

    Article Title: Measuring Nucleosome Assembly Activity in vitro with the Nucleosome Assembly and Quantification (NAQ) Assay
    Article Snippet: Low retention pipette tips (USA Scientific) PCR tubes (USA Scientific, catalog number: 1402-4308) 1.5 ml tubes (Fisher Scientific, catalog number: 05-408-129) 207 bp DNA (procedure explained in ) Loading control DNA [of length between 400 and 1,000 bp, we use a 621 bp DNA (procedure explained in )] Refolded H2A-H2B (procedure explained in ) or H2A-H2B labeled with ATTO 647N (procedure explained in ). .. Refolded (H3-H4)2 (procedure explained in ) Recombinant histone chaperone Salt-assembled nucleosome (procedures explained in ) 50% glycerol (autoclaved and stored at room temperature) 50 bp DNA ladder (Gold Bio, catalog number: D100-500) 10x MNase buffer (New England Biolabs, provided with M0247S) 100x BSA (New England Biolabs, catalog number: B9001) Note: This product has been discontinued.

    Article Title: Nucleosome positioning in the regulatory region of SV40 chromatin correlates with the activation and repression of early and late transcription during infection
    Article Snippet: Following sonication the sample was purified using Zymo Research ChIP DNA Clean and Concentrator columns according to their protocol and eluted in 51 μl H2 O. Micrococcal nuclease digested chromatin was obtained by treating 200 μl of SV40 chromatin with micrococcal nuclease (New England Biolabs, M0247S) at 4 °C. .. Following sonication the sample was purified using Zymo Research ChIP DNA Clean and Concentrator columns according to their protocol and eluted in 51 μl H2 O. Micrococcal nuclease digested chromatin was obtained by treating 200 μl of SV40 chromatin with micrococcal nuclease (New England Biolabs, M0247S) at 4 °C.

    Article Title: Temporal Analysis of the Honey Bee Microbiome Reveals Four Novel Viruses and Seasonal Prevalence of Known Viruses, Nosema, and Crithidia
    Article Snippet: DNA was extracted using the DNeasy Genomic DNA Extraction Kit (Qiagen) as per the manufacturer's instructions. .. Bees from Crithidia positive hives were homogenized by TissueLyser as above and DNA extracted using the DNeasy kit for the initial PCR screens, after suspension in either PBS or 1× Micrococcal Nuclease Buffer (NEB). .. Total nucleic acid from all twenty monitor hives at time-point 17 (August 5, 2009) was pooled (approximately 3 µg per hive).

    Article Title: Single-molecule compaction of megabase-long chromatin molecules by multivalent cations
    Article Snippet: Chromatin was digested by Micrococcal Nuclease (MNase) (New England Biolabs) following established procedure with modifications ( ). .. After the addition of 25 μg of Proteinase K (Thermofisher Scientific), the solution was incubated at 50°C for 30 min.

    Article Title: DNA template strand sequencing of single-cells maps genomic rearrangements at high resolution
    Article Snippet: Single cells were sorted directly into 100 μl lysis buffer (nuclei isolation buffer, NucleiEZ kit, Sigma) in flexible unskirted PCR plates (Bio-Rad) fitted into a rigid plate holder for sorting and spinning. .. Next, 40 μl of 1.25× micrococcal nuclease (MNase) master mix (62.5 mM Tris-HCl pH 7.9, 6.25 mM CaCl, 0.03125 U/μl MNase enzyme, New England Biolabs) was added to each well containing a nucleus (as well as to no-cell negative-control wells containing only lysis buffer).

    Construct:

    Article Title: Determinants of nucleosome positioning and their influence on plant gene expression
    Article Snippet: For isolating nucleosome gDNA, the nuclei were purified from one aliquot with nuclei extraction buffer (10 mM Hepes [pH 7.6], 5 mM potassium chloride, 5 mM magnesium chloride, 1 M sucrose, 14 mM β-mercaptoethanol, 0.6% Triton X-100, 0.4 mM phenylmethanesulfonyl fluoride, 1× ethylenediaminetetraacetic acid-free protease inhibitor [Roche]), digested with 0.4 units/μL micrococcal nuclease (MNase) (NEB) for 9 min, and purified with phenol/chloroform. .. For isolating nucleosome gDNA, the nuclei were purified from one aliquot with nuclei extraction buffer (10 mM Hepes [pH 7.6], 5 mM potassium chloride, 5 mM magnesium chloride, 1 M sucrose, 14 mM β-mercaptoethanol, 0.6% Triton X-100, 0.4 mM phenylmethanesulfonyl fluoride, 1× ethylenediaminetetraacetic acid-free protease inhibitor [Roche]), digested with 0.4 units/μL micrococcal nuclease (MNase) (NEB) for 9 min, and purified with phenol/chloroform.

    Incubation:

    Article Title: ASSAY DEVELOPMENT AND HIGH THROUGHPUT SCREENING FOR INHIBITORS OF KAPOSI'S SARCOMA-ASSOCIATED HERPESVIRUS N-TERMINAL LANA BINDING TO NUCLEOSOMES
    Article Snippet: Flow-through containing RBC nuclei was centrifuged 5 min at 3500xg and pelleted nuclei were washed once with 10 volumes RBC lysis buffer followed by two additional washes (or until the supernatant and pellet were no longer red) with washing buffer (RBC lysis buffer without NP-40). .. Nuclei were then resuspended in micronuclease (MNase) buffer (15mM HEPES pH 7.5, 65mM NaCl, 65mM KCl, 2mM MgCl2 , 5mM CaCl2 and Complete EDTA-Free protease inhibitors cocktail tablet [Roche]) and incubated 2h at 37°C with 800 kunitz units MNase (NEB #M0247S)/10mL MNase buffer, to generate short chromatin of 1-6 nucleosomes long. .. MNase digests linker DNA.

    Article Title: N6-methyladenine is an epigenetic marker of mammalian early life stress
    Article Snippet: Samples were homogenized with a pestle then lightly sonicated via probe-based sonication to fully lyse cells and the nuclear envelope. .. Chromatin shearing was obtained using 1000 gel units of Micrococcal Nuclease (MNase) (New England Biolabs; catalog #M0247S); samples were incubated with Proteinase K to ensure efficient shearing of DNA (between 300 bp–900 bp). .. The MNase reaction was stopped with 1.25 μmol EGTA.

    Article Title: TRF2 is recruited to the pre-initiation complex as a testis-specific subunit of TFIIA/ALF to promote haploid cell gene expression
    Article Snippet: Nuclei were washed once with 1X PBS and spin and resuspended in 2 volumes of MNase lysis buffer (50 mM Tris pH8, 0.5% Na deoxycholate). .. The lysate was sonicated for 30 sec as described above, CaCl2 and MgCl2 were added to 5 mM final concentration each and digestion was performed with MNase (10 gel units/uL final) (M0247S, New England BioLabs) for 10 min of incubation. .. The reaction was stopped by addition of EDTA to 10 mM final concentration.

    Article Title: Characterization of a protein–protein interaction within the SigO–RsoA two-subunit σ factor: the σ70 region 2.3-like segment of RsoA mediates interaction with SigO
    Article Snippet: Transformed cells were grown overnight at 37 °C with shaking and 100 µl of this culture was used to inoculate 5 ml of fresh medium. .. Cell pellets were subjected to two freeze–thaw cycles and the pellets were re-suspended in 200 µl of 10 mM Tris/HCl (pH 8.0), supplemented with 1× micrococcal nuclease buffer and 10 000 U micrococcal nuclease (NEB) and incubated at room temperature for 30 min until cells were lysed and genomic DNA was digested. .. The cell lysate was brought to a volume of 1 ml with binding buffer [20 mM Tris/HCl (pH 7.5), 500 mM NaCl and 0.05 % Tween-20] and a 300 µl volume was retained as crude lysate.

    Article Title: Nonantibiotic Effects of Fluoroquinolones in Mammalian Cells
    Article Snippet: RNase A solution (Thermo Scientific, final concentration 10 mg/ml) and proteinase K solution (Sigma, final concentration 10 mg/ml) were added to the lysed nuclei and incubated overnight at 55 °C. .. The resulting 40-μl mixture contained 3 μg of DNA, 1× micrococcal nuclease buffer (New England Biolabs), 400 mm MgCl2 , 4 mm ZnCl2 , 20 units of deoxyribonuclease I (New England Biolabs), 2000 units of micrococcal nuclease I (New England Biolabs), 5 units of antarctic phosphatase (New England Biolabs), and 0.4 units of snake venom phosphodiesterase.

    Article Title: Single-molecule compaction of megabase-long chromatin molecules by multivalent cations
    Article Snippet: Chromatin was digested by Micrococcal Nuclease (MNase) (New England Biolabs) following established procedure with modifications ( ). .. Digestion was stopped by addition of EDTA to 100 mM final concentration.

    Article Title: CELF RNA binding proteins promote axon regeneration in C. elegans and mammals through alternative splicing of Syntaxins
    Article Snippet: Beads were collected and washed twice with Wash Buffer (1X PBS with 0.1% SDS, 0.5% sodium deoxycholate, and 0.5% NP-40), twice with High Salt Wash Buffer (5X PBS, 0.1% SDS, 0.5% sodium deoxycholate, and 0.5% NP-40) and twice with Polynucleotide Kinase Buffer (PNK Buffer) (50 mM Tris-Cl pH 7.4, 10 mM MgCl2 and 0.5% NP-40). .. Beads were incubated with 500 μl of Micrococcal Nuclease Reaction Buffer (50 mM Tris-Cl pH 7.9, 5 mM CaCl2 ) containing 1 ng of Micrococcal Nuclease (NEB) for a total of 10 min at 4°C with intermittent shaking on a Thermomixer (Eppendorf) (1200 rpm for 1 min and then 1200 rpm for 15 sec every 3 min). .. Beads were then washed twice with PNK+EGTA Buffer (50 mM Tris-Cl pH 7.4, 20 mM EGTA and 0.5% NP-40), twice with Wash Buffer and twice with PNK Buffer.

    Article Title: Transient Downregulation of Nanog and Oct4 Induced by DETA/NO Exposure in Mouse Embryonic Stem Cells Leads to Mesodermal/Endodermal Lineage Differentiation
    Article Snippet: Following centrifugation at 4°C for 5 min, supernatants were discarded and pellets were washed by gentle inversion with 10 mM Tris HCl buffer, pH 8.0 containing 15 mM NaCl, and 60 mM KCl and centrifuged for 5 min at 4°C. .. Pellets were then resuspended in washing buffer supplemented with 3 mM CaCl2 , protease inhibitors, 0.5 mM DTT, and 5–10 μ L of 1 : 200 micrococcal nuclease (New England BioLabs) and incubated for 20 min at 37°C with orbital shaking. .. Nuclease activity was halted by addition of 20 μ L 0.5 mM EDTA and preparations were then centrifuged for 5 min at 3000 rpm.

    Expressing:

    Article Title: Characterization of a protein–protein interaction within the SigO–RsoA two-subunit σ factor: the σ70 region 2.3-like segment of RsoA mediates interaction with SigO
    Article Snippet: Paragraph title: Tandem SigO/RsoA expression and pull-down assays. ... Cell pellets were subjected to two freeze–thaw cycles and the pellets were re-suspended in 200 µl of 10 mM Tris/HCl (pH 8.0), supplemented with 1× micrococcal nuclease buffer and 10 000 U micrococcal nuclease (NEB) and incubated at room temperature for 30 min until cells were lysed and genomic DNA was digested.

    Article Title: Determinants of nucleosome positioning and their influence on plant gene expression
    Article Snippet: For isolating nucleosome gDNA, the nuclei were purified from one aliquot with nuclei extraction buffer (10 mM Hepes [pH 7.6], 5 mM potassium chloride, 5 mM magnesium chloride, 1 M sucrose, 14 mM β-mercaptoethanol, 0.6% Triton X-100, 0.4 mM phenylmethanesulfonyl fluoride, 1× ethylenediaminetetraacetic acid-free protease inhibitor [Roche]), digested with 0.4 units/μL micrococcal nuclease (MNase) (NEB) for 9 min, and purified with phenol/chloroform. .. Instead of cutting the 150 bp DNA fragments from the agarose gels , all the purified DNA fragments were used to construct libraries for 50-bp paired-end (PE) sequencing, following an approach that evaluates not only nucleosome occupancy but also regulator binding sites ( ).

    Cell Fractionation:

    Article Title: Androgen Receptor Serine 81 Phosphorylation Mediates Chromatin Binding and Transcriptional Activation
    Article Snippet: Paragraph title: Cellular Fractionation Assay ... The insoluble fraction (pellet left from cytoplasmic and nuclear protein extraction) was suspended in 40 μl of micrococcal nuclease digestion buffer (M0247, New England Biolabs), kept at room temperature, and treated with 600 units of micrococcal nuclease for 2 min.

    Modification:

    Article Title: CELF RNA binding proteins promote axon regeneration in C. elegans and mammals through alternative splicing of Syntaxins
    Article Snippet: Beads were incubated with 500 μl of Micrococcal Nuclease Reaction Buffer (50 mM Tris-Cl pH 7.9, 5 mM CaCl2 ) containing 1 ng of Micrococcal Nuclease (NEB) for a total of 10 min at 4°C with intermittent shaking on a Thermomixer (Eppendorf) (1200 rpm for 1 min and then 1200 rpm for 15 sec every 3 min). .. Beads were incubated with 500 μl of Micrococcal Nuclease Reaction Buffer (50 mM Tris-Cl pH 7.9, 5 mM CaCl2 ) containing 1 ng of Micrococcal Nuclease (NEB) for a total of 10 min at 4°C with intermittent shaking on a Thermomixer (Eppendorf) (1200 rpm for 1 min and then 1200 rpm for 15 sec every 3 min).

    Transformation Assay:

    Article Title: Characterization of a protein–protein interaction within the SigO–RsoA two-subunit σ factor: the σ70 region 2.3-like segment of RsoA mediates interaction with SigO
    Article Snippet: Cell pellets were subjected to two freeze–thaw cycles and the pellets were re-suspended in 200 µl of 10 mM Tris/HCl (pH 8.0), supplemented with 1× micrococcal nuclease buffer and 10 000 U micrococcal nuclease (NEB) and incubated at room temperature for 30 min until cells were lysed and genomic DNA was digested. .. Cell pellets were subjected to two freeze–thaw cycles and the pellets were re-suspended in 200 µl of 10 mM Tris/HCl (pH 8.0), supplemented with 1× micrococcal nuclease buffer and 10 000 U micrococcal nuclease (NEB) and incubated at room temperature for 30 min until cells were lysed and genomic DNA was digested.

    RNA Binding Assay:

    Article Title: Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor
    Article Snippet: The beads tethering SBP-eIF4A were treated with 1x Micrococcal Nuclease Buffer (NEB), 0.5x lysis buffer, 0.5% Triton X-100, and 200 U/μl Micrococcal Nuclease (NEB) in 30 μl at 25 °C for 30 min, washed 5 times with lysis buffer containing 1% Triton X-100, 1M NaCl, and 5 mM EGTA pH 7.4, and rinsed twice with lysis buffer containing 0.1% Triton X-100. .. SBP-eIF4A/RNA complex was eluted with 30 μl of lysis buffer containing 0.1% Triton X-100, 2 mM AMP-PNP, and 5 mM biotin.

    Flow Cytometry:

    Article Title: ASSAY DEVELOPMENT AND HIGH THROUGHPUT SCREENING FOR INHIBITORS OF KAPOSI'S SARCOMA-ASSOCIATED HERPESVIRUS N-TERMINAL LANA BINDING TO NUCLEOSOMES
    Article Snippet: Flow-through containing RBC nuclei was centrifuged 5 min at 3500xg and pelleted nuclei were washed once with 10 volumes RBC lysis buffer followed by two additional washes (or until the supernatant and pellet were no longer red) with washing buffer (RBC lysis buffer without NP-40). .. Nuclei were then resuspended in micronuclease (MNase) buffer (15mM HEPES pH 7.5, 65mM NaCl, 65mM KCl, 2mM MgCl2 , 5mM CaCl2 and Complete EDTA-Free protease inhibitors cocktail tablet [Roche]) and incubated 2h at 37°C with 800 kunitz units MNase (NEB #M0247S)/10mL MNase buffer, to generate short chromatin of 1-6 nucleosomes long.

    Immunoprecipitation:

    Article Title: Transient Downregulation of Nanog and Oct4 Induced by DETA/NO Exposure in Mouse Embryonic Stem Cells Leads to Mesodermal/Endodermal Lineage Differentiation
    Article Snippet: Pellets were then resuspended in washing buffer supplemented with 3 mM CaCl2 , protease inhibitors, 0.5 mM DTT, and 5–10 μ L of 1 : 200 micrococcal nuclease (New England BioLabs) and incubated for 20 min at 37°C with orbital shaking. .. Supernatants were discarded and pellets were resuspended in buffer containing 150 mM NaCl, 50 mM Tris HCl (pH 7.5), 5 mM EDTA, 0.5% NP-40, 1% Triton, and 0.01% SDS and sonicated with 3 pulses, 10 s each at 10% amplitude.

    Protease Inhibitor:

    Article Title: N6-methyladenine is an epigenetic marker of mammalian early life stress
    Article Snippet: Samples were washed 2x with PBS and cOmplete protease inhibitor (CPI) tablets (Roche, Basel, Switzerland; catalog #04693116001) then resuspended in 400 μL RIPA lysis buffer + CPI tablets. .. Chromatin shearing was obtained using 1000 gel units of Micrococcal Nuclease (MNase) (New England Biolabs; catalog #M0247S); samples were incubated with Proteinase K to ensure efficient shearing of DNA (between 300 bp–900 bp).

    Article Title: Determinants of nucleosome positioning and their influence on plant gene expression
    Article Snippet: The harvested samples were ground with liquid nitrogen and divided into three aliquots for nucleosome genomic DNA (gDNA), naked gDNA, and RNA isolation. .. For isolating nucleosome gDNA, the nuclei were purified from one aliquot with nuclei extraction buffer (10 mM Hepes [pH 7.6], 5 mM potassium chloride, 5 mM magnesium chloride, 1 M sucrose, 14 mM β-mercaptoethanol, 0.6% Triton X-100, 0.4 mM phenylmethanesulfonyl fluoride, 1× ethylenediaminetetraacetic acid-free protease inhibitor [Roche]), digested with 0.4 units/μL micrococcal nuclease (MNase) (NEB) for 9 min, and purified with phenol/chloroform. .. To generate the naked gDNA sample, the gDNA was isolated without the nucleus isolation step as described previously , purified with phenol/chloroform to strip off proteins, and digested with 0.25 units/μL MNase (NEB) for 1.75 or 3 min.

    Article Title: Ectopic T Cell Receptor-? Locus Control Region Activity in B Cells Is Suppressed by Direct Linkage to Two Flanking Genes at Once
    Article Snippet: Thymocytes and B cells of analyzed transgenic mouse lines were fixed in 10 ml RPMI medium with 1% formaldehyde at room temperature for 10 minutes. .. The cells were washed twice with 1× PBS and the cell pellets were re-suspended in 1 ml 1× micrococcal nuclease (MNase) buffer (NEB) containing 2.5 µl protease inhibitor cocktail (Sigma) and 1 µl 100 mM PMSF. .. Nucleosomes were prepared by incubating with 500 units of MNase (NEB) for 10 minutes at 37°C.

    Article Title: Androgen Receptor Serine 81 Phosphorylation Mediates Chromatin Binding and Transcriptional Activation
    Article Snippet: For extraction, LNCaP cells grown in a 10-cm dish were washed once in cold PBS, harvested for resuspension in 800 μl of TLB containing protease inhibitor and phosphatase inhibitor but no salt (NaCl), vortexed, and kept on ice for 15 min, followed by centrifugation at 11,000 × g for 5 min. .. The insoluble fraction (pellet left from cytoplasmic and nuclear protein extraction) was suspended in 40 μl of micrococcal nuclease digestion buffer (M0247, New England Biolabs), kept at room temperature, and treated with 600 units of micrococcal nuclease for 2 min.

    Transferring:

    Article Title: Measuring Nucleosome Assembly Activity in vitro with the Nucleosome Assembly and Quantification (NAQ) Assay
    Article Snippet: Low retention pipette tips (USA Scientific) PCR tubes (USA Scientific, catalog number: 1402-4308) 1.5 ml tubes (Fisher Scientific, catalog number: 05-408-129) 207 bp DNA (procedure explained in ) Loading control DNA [of length between 400 and 1,000 bp, we use a 621 bp DNA (procedure explained in )] Refolded H2A-H2B (procedure explained in ) or H2A-H2B labeled with ATTO 647N (procedure explained in ). .. Refolded (H3-H4)2 (procedure explained in ) Recombinant histone chaperone Salt-assembled nucleosome (procedures explained in ) 50% glycerol (autoclaved and stored at room temperature) 50 bp DNA ladder (Gold Bio, catalog number: D100-500) 10x MNase buffer (New England Biolabs, provided with M0247S) 100x BSA (New England Biolabs, catalog number: B9001) Note: This product has been discontinued.

    Hemagglutination Assay:

    Article Title: Characterization of a protein–protein interaction within the SigO–RsoA two-subunit σ factor: the σ70 region 2.3-like segment of RsoA mediates interaction with SigO
    Article Snippet: The rsoA gene (and mutant derivatives of the gene) was fused to the T25 cyaA gene fragment and an HA epitope was imparted 3′ to rsoA . .. Cell pellets were subjected to two freeze–thaw cycles and the pellets were re-suspended in 200 µl of 10 mM Tris/HCl (pH 8.0), supplemented with 1× micrococcal nuclease buffer and 10 000 U micrococcal nuclease (NEB) and incubated at room temperature for 30 min until cells were lysed and genomic DNA was digested.

    Light Microscopy:

    Article Title: Temporal Analysis of the Honey Bee Microbiome Reveals Four Novel Viruses and Seasonal Prevalence of Known Viruses, Nosema, and Crithidia
    Article Snippet: Light microscopy of live parasites was performed using a Leica DM6000 microscope equipped with Hamamatsu C4742-95 camera and Volocity Software (PerkinElmer). .. Bees from Crithidia positive hives were homogenized by TissueLyser as above and DNA extracted using the DNeasy kit for the initial PCR screens, after suspension in either PBS or 1× Micrococcal Nuclease Buffer (NEB).

    Imaging:

    Article Title: Temporal Analysis of the Honey Bee Microbiome Reveals Four Novel Viruses and Seasonal Prevalence of Known Viruses, Nosema, and Crithidia
    Article Snippet: Imaging fixed parasites (4% paraformaldehyde, 20 min) facilitated visualization of DAPI (4′,6-diamidino-2-phenylindole) stained nuclear and kinetoplast DNA. .. Bees from Crithidia positive hives were homogenized by TissueLyser as above and DNA extracted using the DNeasy kit for the initial PCR screens, after suspension in either PBS or 1× Micrococcal Nuclease Buffer (NEB).

    Sequencing:

    Article Title: Nucleosome positioning in the regulatory region of SV40 chromatin correlates with the activation and repression of early and late transcription during infection
    Article Snippet: Paragraph title: 4.4. Preparation of sequencing libraries from SV40 chromatin fragmented by either sonication or micrococcal nuclease digestion ... Following sonication the sample was purified using Zymo Research ChIP DNA Clean and Concentrator columns according to their protocol and eluted in 51 μl H2 O. Micrococcal nuclease digested chromatin was obtained by treating 200 μl of SV40 chromatin with micrococcal nuclease (New England Biolabs, M0247S) at 4 °C.

    Article Title: Determinants of nucleosome positioning and their influence on plant gene expression
    Article Snippet: For isolating nucleosome gDNA, the nuclei were purified from one aliquot with nuclei extraction buffer (10 mM Hepes [pH 7.6], 5 mM potassium chloride, 5 mM magnesium chloride, 1 M sucrose, 14 mM β-mercaptoethanol, 0.6% Triton X-100, 0.4 mM phenylmethanesulfonyl fluoride, 1× ethylenediaminetetraacetic acid-free protease inhibitor [Roche]), digested with 0.4 units/μL micrococcal nuclease (MNase) (NEB) for 9 min, and purified with phenol/chloroform. .. For isolating nucleosome gDNA, the nuclei were purified from one aliquot with nuclei extraction buffer (10 mM Hepes [pH 7.6], 5 mM potassium chloride, 5 mM magnesium chloride, 1 M sucrose, 14 mM β-mercaptoethanol, 0.6% Triton X-100, 0.4 mM phenylmethanesulfonyl fluoride, 1× ethylenediaminetetraacetic acid-free protease inhibitor [Roche]), digested with 0.4 units/μL micrococcal nuclease (MNase) (NEB) for 9 min, and purified with phenol/chloroform.

    Article Title: CELF RNA binding proteins promote axon regeneration in C. elegans and mammals through alternative splicing of Syntaxins
    Article Snippet: Paragraph title: Crosslinking Immunoprecipitation and deep sequencing (CLIP-seq) in C. elegans ... Beads were incubated with 500 μl of Micrococcal Nuclease Reaction Buffer (50 mM Tris-Cl pH 7.9, 5 mM CaCl2 ) containing 1 ng of Micrococcal Nuclease (NEB) for a total of 10 min at 4°C with intermittent shaking on a Thermomixer (Eppendorf) (1200 rpm for 1 min and then 1200 rpm for 15 sec every 3 min).

    Sonication:

    Article Title: N6-methyladenine is an epigenetic marker of mammalian early life stress
    Article Snippet: Samples were homogenized with a pestle then lightly sonicated via probe-based sonication to fully lyse cells and the nuclear envelope. .. Chromatin shearing was obtained using 1000 gel units of Micrococcal Nuclease (MNase) (New England Biolabs; catalog #M0247S); samples were incubated with Proteinase K to ensure efficient shearing of DNA (between 300 bp–900 bp).

    Article Title: TRF2 is recruited to the pre-initiation complex as a testis-specific subunit of TFIIA/ALF to promote haploid cell gene expression
    Article Snippet: Nuclei were washed once with 1X PBS and spin and resuspended in 2 volumes of MNase lysis buffer (50 mM Tris pH8, 0.5% Na deoxycholate). .. The lysate was sonicated for 30 sec as described above, CaCl2 and MgCl2 were added to 5 mM final concentration each and digestion was performed with MNase (10 gel units/uL final) (M0247S, New England BioLabs) for 10 min of incubation. .. The reaction was stopped by addition of EDTA to 10 mM final concentration.

    Article Title: Nucleosome positioning in the regulatory region of SV40 chromatin correlates with the activation and repression of early and late transcription during infection
    Article Snippet: The sample (200 μl) in an Eppendorf tube was placed in a cup containing cold flowing water for cooling. .. Following sonication the sample was purified using Zymo Research ChIP DNA Clean and Concentrator columns according to their protocol and eluted in 51 μl H2 O. Micrococcal nuclease digested chromatin was obtained by treating 200 μl of SV40 chromatin with micrococcal nuclease (New England Biolabs, M0247S) at 4 °C. .. Following digestion, the DNA was purified as described for sonicated DNA.

    Article Title: CELF RNA binding proteins promote axon regeneration in C. elegans and mammals through alternative splicing of Syntaxins
    Article Snippet: Worms were lysed by sonication in Homogenization Buffer (100 mM NaCl, 25 mM HEPES, 250 μM EDTA, 2 mM DTT, 0.1% NP-40, 25 units/ml RNasin and Protease Inhibitors). .. Beads were incubated with 500 μl of Micrococcal Nuclease Reaction Buffer (50 mM Tris-Cl pH 7.9, 5 mM CaCl2 ) containing 1 ng of Micrococcal Nuclease (NEB) for a total of 10 min at 4°C with intermittent shaking on a Thermomixer (Eppendorf) (1200 rpm for 1 min and then 1200 rpm for 15 sec every 3 min).

    Article Title: Transient Downregulation of Nanog and Oct4 Induced by DETA/NO Exposure in Mouse Embryonic Stem Cells Leads to Mesodermal/Endodermal Lineage Differentiation
    Article Snippet: Pellets were then resuspended in washing buffer supplemented with 3 mM CaCl2 , protease inhibitors, 0.5 mM DTT, and 5–10 μ L of 1 : 200 micrococcal nuclease (New England BioLabs) and incubated for 20 min at 37°C with orbital shaking. .. Nuclease activity was halted by addition of 20 μ L 0.5 mM EDTA and preparations were then centrifuged for 5 min at 3000 rpm.

    Recombinant:

    Article Title: Measuring Nucleosome Assembly Activity in vitro with the Nucleosome Assembly and Quantification (NAQ) Assay
    Article Snippet: Low retention pipette tips (USA Scientific) PCR tubes (USA Scientific, catalog number: 1402-4308) 1.5 ml tubes (Fisher Scientific, catalog number: 05-408-129) 207 bp DNA (procedure explained in ) Loading control DNA [of length between 400 and 1,000 bp, we use a 621 bp DNA (procedure explained in )] Refolded H2A-H2B (procedure explained in ) or H2A-H2B labeled with ATTO 647N (procedure explained in ). .. Refolded (H3-H4)2 (procedure explained in ) Recombinant histone chaperone Salt-assembled nucleosome (procedures explained in ) 50% glycerol (autoclaved and stored at room temperature) 50 bp DNA ladder (Gold Bio, catalog number: D100-500) 10x MNase buffer (New England Biolabs, provided with M0247S) 100x BSA (New England Biolabs, catalog number: B9001) Note: This product has been discontinued. .. Micrococcal nuclease (MNase) (New England Biolabs, catalog number: M0247S) MinElute kit (QIAGEN, catalog number: 28006) Proteinase K, 20 mg/ml solution (BioExpress, catalog number: E195-5ML) Tris (2-Carboxyethyl) phosphine Hydrochloride (TCEP) (Gold Bio, catalog number: TCEP100) Sodium hydroxide (NaOH) (Fisher Scientific, catalog number: S318-3) Tris base (Fisher Scientific, catalog number: BP152-5) Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-10) 500 mM EDTA solution at pH ~8 (stored at room temperature) Tween-20 (Fisher Scientific, catalog number: BP337) SYBR Gold nucleic acid gel stain (Thermo Fisher Scientific, Invitrogen™ , catalog number: S11494) Boric acid (Acros Organics, catalog number: 180570025) Ammonium persulfate (AMRESCO, catalog number: 0486) 30% acrylamide 37.5:1 (Life science Products, catalog number: EC-890) Tetramethylethylenediamine (TEMED) (Fisher Scientific, catalog number: BP150-20) Bromophenol blue (Fisher Scientific, catalog number: B392-5) Xylene cyanol FF (Sigma Aldrich, catalog number: X4126) Sodium acetate (Fisher Scientific, catalog number: S210-500) Acetic acid (Avantor Performance Materials, catalog number: V193-46) 1 M TCEP (Tris (2-Carboxyethyl) phosphine Hydrochloride; see Recipes) NA buffer (see Recipes) SYBR Gold stain solution (see Recipes) 10x TBE (Tris/Borate/EDTA; see Recipes) 25% APS (Ammonium Persulfate; see Recipes) 6% PAGE gels (see Recipes) 10% PAGE gels (see Recipes) DNA sample buffer (see Recipes) 3 M Na acetate pH 5.0 solution (see Recipes)

    Staining:

    Article Title: The ATP-dependent chromatin remodeling enzymes CHD6, CHD7, and CHD8 exhibit distinct nucleosome binding and remodeling activities
    Article Snippet: After 30 min, 1.5 mm final CaCl2 and 1 gel unit of MNase (New England Biolabs, M0247S) were supplemented, and the reaction proceeded at 37 °C for 10 min. .. DNA was immediately mixed with loading dye and subjected to 0.5× TBE 4% (29:1) native PAGE at 100 V for 40 min.

    Article Title: Temporal Analysis of the Honey Bee Microbiome Reveals Four Novel Viruses and Seasonal Prevalence of Known Viruses, Nosema, and Crithidia
    Article Snippet: Imaging fixed parasites (4% paraformaldehyde, 20 min) facilitated visualization of DAPI (4′,6-diamidino-2-phenylindole) stained nuclear and kinetoplast DNA. .. Bees from Crithidia positive hives were homogenized by TissueLyser as above and DNA extracted using the DNeasy kit for the initial PCR screens, after suspension in either PBS or 1× Micrococcal Nuclease Buffer (NEB).

    DNA Extraction:

    Article Title: Temporal Analysis of the Honey Bee Microbiome Reveals Four Novel Viruses and Seasonal Prevalence of Known Viruses, Nosema, and Crithidia
    Article Snippet: DNA was extracted using the DNeasy Genomic DNA Extraction Kit (Qiagen) as per the manufacturer's instructions. .. Bees from Crithidia positive hives were homogenized by TissueLyser as above and DNA extracted using the DNeasy kit for the initial PCR screens, after suspension in either PBS or 1× Micrococcal Nuclease Buffer (NEB).

    Article Title: Nonantibiotic Effects of Fluoroquinolones in Mammalian Cells
    Article Snippet: Paragraph title: Genomic DNA Extraction and Hydrolysis ... The resulting 40-μl mixture contained 3 μg of DNA, 1× micrococcal nuclease buffer (New England Biolabs), 400 mm MgCl2 , 4 mm ZnCl2 , 20 units of deoxyribonuclease I (New England Biolabs), 2000 units of micrococcal nuclease I (New England Biolabs), 5 units of antarctic phosphatase (New England Biolabs), and 0.4 units of snake venom phosphodiesterase.

    DNA Purification:

    Article Title: Temporal Analysis of the Honey Bee Microbiome Reveals Four Novel Viruses and Seasonal Prevalence of Known Viruses, Nosema, and Crithidia
    Article Snippet: Paragraph title: Crithidia mellificae strain SF - Microscopy, Culturing and DNA Purification ... Bees from Crithidia positive hives were homogenized by TissueLyser as above and DNA extracted using the DNeasy kit for the initial PCR screens, after suspension in either PBS or 1× Micrococcal Nuclease Buffer (NEB).

    Methylation:

    Article Title: Planarian MBD2/3 is required for adult stem cell pluripotency independently of DNA methylation
    Article Snippet: Genomic DNA was prepared from S. mediterranea , which had been starved for at least 2 weeks weeks to minimize any potential dietary source of methylated DNA. .. Each 20 ml reaction contained S. mediterranea DNA, 5 units of Antarctic Phosphatase (NEB) and 2 mU Snake venom Phosphodiesterase (Phosphodiesterase I from Crotalus adamentus venom; Sigma) in a buffer that was 0.5×Micrococcal Nuclease Buffer (NEB) and 0.5×Antarctic Phosphatase Buffer (NEB).

    Mutagenesis:

    Article Title: Characterization of a protein–protein interaction within the SigO–RsoA two-subunit σ factor: the σ70 region 2.3-like segment of RsoA mediates interaction with SigO
    Article Snippet: The rsoA gene (and mutant derivatives of the gene) was fused to the T25 cyaA gene fragment and an HA epitope was imparted 3′ to rsoA . .. Cell pellets were subjected to two freeze–thaw cycles and the pellets were re-suspended in 200 µl of 10 mM Tris/HCl (pH 8.0), supplemented with 1× micrococcal nuclease buffer and 10 000 U micrococcal nuclease (NEB) and incubated at room temperature for 30 min until cells were lysed and genomic DNA was digested.

    Isolation:

    Article Title: ASSAY DEVELOPMENT AND HIGH THROUGHPUT SCREENING FOR INHIBITORS OF KAPOSI'S SARCOMA-ASSOCIATED HERPESVIRUS N-TERMINAL LANA BINDING TO NUCLEOSOMES
    Article Snippet: Erythrocytes were isolated immediately when received by washing three times (or until no more thin cream-colored layer of white cells was observed over the red cells) with PBS containing 5% citrate (to prevent coagulation). .. Nuclei were then resuspended in micronuclease (MNase) buffer (15mM HEPES pH 7.5, 65mM NaCl, 65mM KCl, 2mM MgCl2 , 5mM CaCl2 and Complete EDTA-Free protease inhibitors cocktail tablet [Roche]) and incubated 2h at 37°C with 800 kunitz units MNase (NEB #M0247S)/10mL MNase buffer, to generate short chromatin of 1-6 nucleosomes long.

    Article Title: Determinants of nucleosome positioning and their influence on plant gene expression
    Article Snippet: Paragraph title: Plant materials and isolation of DNA and RNA ... For isolating nucleosome gDNA, the nuclei were purified from one aliquot with nuclei extraction buffer (10 mM Hepes [pH 7.6], 5 mM potassium chloride, 5 mM magnesium chloride, 1 M sucrose, 14 mM β-mercaptoethanol, 0.6% Triton X-100, 0.4 mM phenylmethanesulfonyl fluoride, 1× ethylenediaminetetraacetic acid-free protease inhibitor [Roche]), digested with 0.4 units/μL micrococcal nuclease (MNase) (NEB) for 9 min, and purified with phenol/chloroform.

    Article Title: DNA template strand sequencing of single-cells maps genomic rearrangements at high resolution
    Article Snippet: Single cells were sorted directly into 100 μl lysis buffer (nuclei isolation buffer, NucleiEZ kit, Sigma) in flexible unskirted PCR plates (Bio-Rad) fitted into a rigid plate holder for sorting and spinning. .. Next, 40 μl of 1.25× micrococcal nuclease (MNase) master mix (62.5 mM Tris-HCl pH 7.9, 6.25 mM CaCl, 0.03125 U/μl MNase enzyme, New England Biolabs) was added to each well containing a nucleus (as well as to no-cell negative-control wells containing only lysis buffer).

    Magnetic Beads:

    Article Title: N6-methyladenine is an epigenetic marker of mammalian early life stress
    Article Snippet: Chromatin shearing was obtained using 1000 gel units of Micrococcal Nuclease (MNase) (New England Biolabs; catalog #M0247S); samples were incubated with Proteinase K to ensure efficient shearing of DNA (between 300 bp–900 bp). .. Chromatin shearing was obtained using 1000 gel units of Micrococcal Nuclease (MNase) (New England Biolabs; catalog #M0247S); samples were incubated with Proteinase K to ensure efficient shearing of DNA (between 300 bp–900 bp).

    Article Title: Characterization of a protein–protein interaction within the SigO–RsoA two-subunit σ factor: the σ70 region 2.3-like segment of RsoA mediates interaction with SigO
    Article Snippet: Cell pellets were subjected to two freeze–thaw cycles and the pellets were re-suspended in 200 µl of 10 mM Tris/HCl (pH 8.0), supplemented with 1× micrococcal nuclease buffer and 10 000 U micrococcal nuclease (NEB) and incubated at room temperature for 30 min until cells were lysed and genomic DNA was digested. .. The cell lysate was brought to a volume of 1 ml with binding buffer [20 mM Tris/HCl (pH 7.5), 500 mM NaCl and 0.05 % Tween-20] and a 300 µl volume was retained as crude lysate.

    Article Title: CELF RNA binding proteins promote axon regeneration in C. elegans and mammals through alternative splicing of Syntaxins
    Article Snippet: Lysates were centrifuged at 16,000g for 15 min at 4°C and supernatants collected and incubated with M2 magnetic beads (Sigma) overnight on a rotator. .. Beads were incubated with 500 μl of Micrococcal Nuclease Reaction Buffer (50 mM Tris-Cl pH 7.9, 5 mM CaCl2 ) containing 1 ng of Micrococcal Nuclease (NEB) for a total of 10 min at 4°C with intermittent shaking on a Thermomixer (Eppendorf) (1200 rpm for 1 min and then 1200 rpm for 15 sec every 3 min).

    Size-exclusion Chromatography:

    Article Title: TRF2 is recruited to the pre-initiation complex as a testis-specific subunit of TFIIA/ALF to promote haploid cell gene expression
    Article Snippet: Nuclei were washed once with 1X PBS and spin and resuspended in 2 volumes of MNase lysis buffer (50 mM Tris pH8, 0.5% Na deoxycholate). .. The lysate was sonicated for 30 sec as described above, CaCl2 and MgCl2 were added to 5 mM final concentration each and digestion was performed with MNase (10 gel units/uL final) (M0247S, New England BioLabs) for 10 min of incubation. .. The reaction was stopped by addition of EDTA to 10 mM final concentration.

    Article Title: CELF RNA binding proteins promote axon regeneration in C. elegans and mammals through alternative splicing of Syntaxins
    Article Snippet: Beads were collected and washed twice with Wash Buffer (1X PBS with 0.1% SDS, 0.5% sodium deoxycholate, and 0.5% NP-40), twice with High Salt Wash Buffer (5X PBS, 0.1% SDS, 0.5% sodium deoxycholate, and 0.5% NP-40) and twice with Polynucleotide Kinase Buffer (PNK Buffer) (50 mM Tris-Cl pH 7.4, 10 mM MgCl2 and 0.5% NP-40). .. Beads were incubated with 500 μl of Micrococcal Nuclease Reaction Buffer (50 mM Tris-Cl pH 7.9, 5 mM CaCl2 ) containing 1 ng of Micrococcal Nuclease (NEB) for a total of 10 min at 4°C with intermittent shaking on a Thermomixer (Eppendorf) (1200 rpm for 1 min and then 1200 rpm for 15 sec every 3 min). .. Beads were then washed twice with PNK+EGTA Buffer (50 mM Tris-Cl pH 7.4, 20 mM EGTA and 0.5% NP-40), twice with Wash Buffer and twice with PNK Buffer.

    Labeling:

    Article Title: Measuring Nucleosome Assembly Activity in vitro with the Nucleosome Assembly and Quantification (NAQ) Assay
    Article Snippet: Low retention pipette tips (USA Scientific) PCR tubes (USA Scientific, catalog number: 1402-4308) 1.5 ml tubes (Fisher Scientific, catalog number: 05-408-129) 207 bp DNA (procedure explained in ) Loading control DNA [of length between 400 and 1,000 bp, we use a 621 bp DNA (procedure explained in )] Refolded H2A-H2B (procedure explained in ) or H2A-H2B labeled with ATTO 647N (procedure explained in ). .. Refolded (H3-H4)2 (procedure explained in ) Recombinant histone chaperone Salt-assembled nucleosome (procedures explained in ) 50% glycerol (autoclaved and stored at room temperature) 50 bp DNA ladder (Gold Bio, catalog number: D100-500) 10x MNase buffer (New England Biolabs, provided with M0247S) 100x BSA (New England Biolabs, catalog number: B9001) Note: This product has been discontinued.

    Article Title: Neutrophil accumulation and NET release contribute to thrombosis in HIT
    Article Snippet: KKO was labeled with Alexa Fluor 647 (Thermo Fisher Scientific) prior to NET channel infusion. .. Similar studies were done using 100 U/ml bacterial-derived micrococcal nuclease (New England Biolabs).

    Purification:

    Article Title: ASSAY DEVELOPMENT AND HIGH THROUGHPUT SCREENING FOR INHIBITORS OF KAPOSI'S SARCOMA-ASSOCIATED HERPESVIRUS N-TERMINAL LANA BINDING TO NUCLEOSOMES
    Article Snippet: Paragraph title: Nucleosome purification from chicken erythrocytes ... Nuclei were then resuspended in micronuclease (MNase) buffer (15mM HEPES pH 7.5, 65mM NaCl, 65mM KCl, 2mM MgCl2 , 5mM CaCl2 and Complete EDTA-Free protease inhibitors cocktail tablet [Roche]) and incubated 2h at 37°C with 800 kunitz units MNase (NEB #M0247S)/10mL MNase buffer, to generate short chromatin of 1-6 nucleosomes long.

    Article Title: The ATP-dependent chromatin remodeling enzymes CHD6, CHD7, and CHD8 exhibit distinct nucleosome binding and remodeling activities
    Article Snippet: 200 ng of chromatinized pGIE-0 plasmid in MNase buffer (20 mm Tris (pH 8.0), 50 mm NaCl, 5 mm MgCl2 , 5% v/v glycerol, 1 mm DTT, 0.1% v/v Tween 20, and 1 mm ATP) was mixed with purified CHD enzyme in MNase buffer on ice, and then the reaction (10 μl) was shifted to 30 °C. .. After 30 min, 1.5 mm final CaCl2 and 1 gel unit of MNase (New England Biolabs, M0247S) were supplemented, and the reaction proceeded at 37 °C for 10 min.

    Article Title: Nucleosome positioning in the regulatory region of SV40 chromatin correlates with the activation and repression of early and late transcription during infection
    Article Snippet: The sample (200 μl) in an Eppendorf tube was placed in a cup containing cold flowing water for cooling. .. Following sonication the sample was purified using Zymo Research ChIP DNA Clean and Concentrator columns according to their protocol and eluted in 51 μl H2 O. Micrococcal nuclease digested chromatin was obtained by treating 200 μl of SV40 chromatin with micrococcal nuclease (New England Biolabs, M0247S) at 4 °C. .. Following digestion, the DNA was purified as described for sonicated DNA.

    Article Title: Nonantibiotic Effects of Fluoroquinolones in Mammalian Cells
    Article Snippet: The resulting 40-μl mixture contained 3 μg of DNA, 1× micrococcal nuclease buffer (New England Biolabs), 400 mm MgCl2 , 4 mm ZnCl2 , 20 units of deoxyribonuclease I (New England Biolabs), 2000 units of micrococcal nuclease I (New England Biolabs), 5 units of antarctic phosphatase (New England Biolabs), and 0.4 units of snake venom phosphodiesterase. .. The resulting 40-μl mixture contained 3 μg of DNA, 1× micrococcal nuclease buffer (New England Biolabs), 400 mm MgCl2 , 4 mm ZnCl2 , 20 units of deoxyribonuclease I (New England Biolabs), 2000 units of micrococcal nuclease I (New England Biolabs), 5 units of antarctic phosphatase (New England Biolabs), and 0.4 units of snake venom phosphodiesterase.

    Article Title: Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor
    Article Snippet: SBP-tagged eIF4A was purified as described in “RIP-Seq”, without DMSO or RocA treatment. .. The beads tethering SBP-eIF4A were treated with 1x Micrococcal Nuclease Buffer (NEB), 0.5x lysis buffer, 0.5% Triton X-100, and 200 U/μl Micrococcal Nuclease (NEB) in 30 μl at 25 °C for 30 min, washed 5 times with lysis buffer containing 1% Triton X-100, 1M NaCl, and 5 mM EGTA pH 7.4, and rinsed twice with lysis buffer containing 0.1% Triton X-100.

    Article Title: Single-molecule compaction of megabase-long chromatin molecules by multivalent cations
    Article Snippet: Chromatin was digested by Micrococcal Nuclease (MNase) (New England Biolabs) following established procedure with modifications ( ). .. After the addition of 25 μg of Proteinase K (Thermofisher Scientific), the solution was incubated at 50°C for 30 min.

    Article Title: Determinants of nucleosome positioning and their influence on plant gene expression
    Article Snippet: The harvested samples were ground with liquid nitrogen and divided into three aliquots for nucleosome genomic DNA (gDNA), naked gDNA, and RNA isolation. .. For isolating nucleosome gDNA, the nuclei were purified from one aliquot with nuclei extraction buffer (10 mM Hepes [pH 7.6], 5 mM potassium chloride, 5 mM magnesium chloride, 1 M sucrose, 14 mM β-mercaptoethanol, 0.6% Triton X-100, 0.4 mM phenylmethanesulfonyl fluoride, 1× ethylenediaminetetraacetic acid-free protease inhibitor [Roche]), digested with 0.4 units/μL micrococcal nuclease (MNase) (NEB) for 9 min, and purified with phenol/chloroform. .. To generate the naked gDNA sample, the gDNA was isolated without the nucleus isolation step as described previously , purified with phenol/chloroform to strip off proteins, and digested with 0.25 units/μL MNase (NEB) for 1.75 or 3 min.

    Article Title: Planarian MBD2/3 is required for adult stem cell pluripotency independently of DNA methylation
    Article Snippet: Up to 5 mg of purified DNA was digested to nucleosides for subsequent LC–MS analysis. .. Each 20 ml reaction contained S. mediterranea DNA, 5 units of Antarctic Phosphatase (NEB) and 2 mU Snake venom Phosphodiesterase (Phosphodiesterase I from Crotalus adamentus venom; Sigma) in a buffer that was 0.5×Micrococcal Nuclease Buffer (NEB) and 0.5×Antarctic Phosphatase Buffer (NEB).

    Transgenic Assay:

    Article Title: Ectopic T Cell Receptor-? Locus Control Region Activity in B Cells Is Suppressed by Direct Linkage to Two Flanking Genes at Once
    Article Snippet: Thymocytes and B cells of analyzed transgenic mouse lines were fixed in 10 ml RPMI medium with 1% formaldehyde at room temperature for 10 minutes. .. The cells were washed twice with 1× PBS and the cell pellets were re-suspended in 1 ml 1× micrococcal nuclease (MNase) buffer (NEB) containing 2.5 µl protease inhibitor cocktail (Sigma) and 1 µl 100 mM PMSF.

    Protein Extraction:

    Article Title: Androgen Receptor Serine 81 Phosphorylation Mediates Chromatin Binding and Transcriptional Activation
    Article Snippet: The micrococcal nuclease digestion was performed as follows. .. The insoluble fraction (pellet left from cytoplasmic and nuclear protein extraction) was suspended in 40 μl of micrococcal nuclease digestion buffer (M0247, New England Biolabs), kept at room temperature, and treated with 600 units of micrococcal nuclease for 2 min. .. The reaction was stopped by adding EGTA (5 m m , final concentration), followed by centrifugation at 11,000 × g for 5 min.

    Coagulation:

    Article Title: ASSAY DEVELOPMENT AND HIGH THROUGHPUT SCREENING FOR INHIBITORS OF KAPOSI'S SARCOMA-ASSOCIATED HERPESVIRUS N-TERMINAL LANA BINDING TO NUCLEOSOMES
    Article Snippet: Erythrocytes were isolated immediately when received by washing three times (or until no more thin cream-colored layer of white cells was observed over the red cells) with PBS containing 5% citrate (to prevent coagulation). .. Nuclei were then resuspended in micronuclease (MNase) buffer (15mM HEPES pH 7.5, 65mM NaCl, 65mM KCl, 2mM MgCl2 , 5mM CaCl2 and Complete EDTA-Free protease inhibitors cocktail tablet [Roche]) and incubated 2h at 37°C with 800 kunitz units MNase (NEB #M0247S)/10mL MNase buffer, to generate short chromatin of 1-6 nucleosomes long.

    Microscopy:

    Article Title: Temporal Analysis of the Honey Bee Microbiome Reveals Four Novel Viruses and Seasonal Prevalence of Known Viruses, Nosema, and Crithidia
    Article Snippet: Paragraph title: Crithidia mellificae strain SF - Microscopy, Culturing and DNA Purification ... Bees from Crithidia positive hives were homogenized by TissueLyser as above and DNA extracted using the DNeasy kit for the initial PCR screens, after suspension in either PBS or 1× Micrococcal Nuclease Buffer (NEB).

    Article Title: Neutrophil accumulation and NET release contribute to thrombosis in HIT
    Article Snippet: NET complexes were imaged with a Zeiss LSM 710 laser-scanning confocal microscope. .. Similar studies were done using 100 U/ml bacterial-derived micrococcal nuclease (New England Biolabs).

    Polyacrylamide Gel Electrophoresis:

    Article Title: Single-molecule compaction of megabase-long chromatin molecules by multivalent cations
    Article Snippet: Chromatin was digested by Micrococcal Nuclease (MNase) (New England Biolabs) following established procedure with modifications ( ). .. After the addition of 25 μg of Proteinase K (Thermofisher Scientific), the solution was incubated at 50°C for 30 min.

    Nuclease Assay:

    Article Title: CHD1L Regulated PARP1‐Driven Pluripotency and Chromatin Remodeling During the Early‐Stage Cell Reprogramming
    Article Snippet: Paragraph title: Micrococcal Nuclease Assay ... Permeabilized cells were then exposed to micrococcal nuclease (MNase) (NEB, MA) at 37 °C for various lengths of time.

    Concentration Assay:

    Article Title: TRF2 is recruited to the pre-initiation complex as a testis-specific subunit of TFIIA/ALF to promote haploid cell gene expression
    Article Snippet: Nuclei were washed once with 1X PBS and spin and resuspended in 2 volumes of MNase lysis buffer (50 mM Tris pH8, 0.5% Na deoxycholate). .. The lysate was sonicated for 30 sec as described above, CaCl2 and MgCl2 were added to 5 mM final concentration each and digestion was performed with MNase (10 gel units/uL final) (M0247S, New England BioLabs) for 10 min of incubation. .. The reaction was stopped by addition of EDTA to 10 mM final concentration.

    Article Title: Nonantibiotic Effects of Fluoroquinolones in Mammalian Cells
    Article Snippet: RNase A solution (Thermo Scientific, final concentration 10 mg/ml) and proteinase K solution (Sigma, final concentration 10 mg/ml) were added to the lysed nuclei and incubated overnight at 55 °C. .. The resulting 40-μl mixture contained 3 μg of DNA, 1× micrococcal nuclease buffer (New England Biolabs), 400 mm MgCl2 , 4 mm ZnCl2 , 20 units of deoxyribonuclease I (New England Biolabs), 2000 units of micrococcal nuclease I (New England Biolabs), 5 units of antarctic phosphatase (New England Biolabs), and 0.4 units of snake venom phosphodiesterase.

    Article Title: Single-molecule compaction of megabase-long chromatin molecules by multivalent cations
    Article Snippet: Chromatin was digested by Micrococcal Nuclease (MNase) (New England Biolabs) following established procedure with modifications ( ). .. Chromatin was digested by Micrococcal Nuclease (MNase) (New England Biolabs) following established procedure with modifications ( ).

    Chromatin Immunoprecipitation:

    Article Title: N6-methyladenine is an epigenetic marker of mammalian early life stress
    Article Snippet: Paragraph title: Chromatin Immunoprecipitation (ChIP) ... Chromatin shearing was obtained using 1000 gel units of Micrococcal Nuclease (MNase) (New England Biolabs; catalog #M0247S); samples were incubated with Proteinase K to ensure efficient shearing of DNA (between 300 bp–900 bp).

    Article Title: Nucleosome positioning in the regulatory region of SV40 chromatin correlates with the activation and repression of early and late transcription during infection
    Article Snippet: The sample (200 μl) in an Eppendorf tube was placed in a cup containing cold flowing water for cooling. .. Following sonication the sample was purified using Zymo Research ChIP DNA Clean and Concentrator columns according to their protocol and eluted in 51 μl H2 O. Micrococcal nuclease digested chromatin was obtained by treating 200 μl of SV40 chromatin with micrococcal nuclease (New England Biolabs, M0247S) at 4 °C. .. Following digestion, the DNA was purified as described for sonicated DNA.

    Article Title: Ectopic T Cell Receptor-? Locus Control Region Activity in B Cells Is Suppressed by Direct Linkage to Two Flanking Genes at Once
    Article Snippet: Paragraph title: Chromatin Immunoprecipitation (ChIP) ... The cells were washed twice with 1× PBS and the cell pellets were re-suspended in 1 ml 1× micrococcal nuclease (MNase) buffer (NEB) containing 2.5 µl protease inhibitor cocktail (Sigma) and 1 µl 100 mM PMSF.

    Article Title: Transient Downregulation of Nanog and Oct4 Induced by DETA/NO Exposure in Mouse Embryonic Stem Cells Leads to Mesodermal/Endodermal Lineage Differentiation
    Article Snippet: Paragraph title: 2.5. Chromatin Immunoprecipitation Assay ... Pellets were then resuspended in washing buffer supplemented with 3 mM CaCl2 , protease inhibitors, 0.5 mM DTT, and 5–10 μ L of 1 : 200 micrococcal nuclease (New England BioLabs) and incubated for 20 min at 37°C with orbital shaking.

    Plasmid Preparation:

    Article Title: The ATP-dependent chromatin remodeling enzymes CHD6, CHD7, and CHD8 exhibit distinct nucleosome binding and remodeling activities
    Article Snippet: 200 ng of chromatinized pGIE-0 plasmid in MNase buffer (20 mm Tris (pH 8.0), 50 mm NaCl, 5 mm MgCl2 , 5% v/v glycerol, 1 mm DTT, 0.1% v/v Tween 20, and 1 mm ATP) was mixed with purified CHD enzyme in MNase buffer on ice, and then the reaction (10 μl) was shifted to 30 °C. .. After 30 min, 1.5 mm final CaCl2 and 1 gel unit of MNase (New England Biolabs, M0247S) were supplemented, and the reaction proceeded at 37 °C for 10 min.

    Article Title: Characterization of a protein–protein interaction within the SigO–RsoA two-subunit σ factor: the σ70 region 2.3-like segment of RsoA mediates interaction with SigO
    Article Snippet: Relevant plasmid pairs were co-transformed into E. coli BL21 carrying pLysS. .. Cell pellets were subjected to two freeze–thaw cycles and the pellets were re-suspended in 200 µl of 10 mM Tris/HCl (pH 8.0), supplemented with 1× micrococcal nuclease buffer and 10 000 U micrococcal nuclease (NEB) and incubated at room temperature for 30 min until cells were lysed and genomic DNA was digested.

    Software:

    Article Title: The ATP-dependent chromatin remodeling enzymes CHD6, CHD7, and CHD8 exhibit distinct nucleosome binding and remodeling activities
    Article Snippet: After 30 min, 1.5 mm final CaCl2 and 1 gel unit of MNase (New England Biolabs, M0247S) were supplemented, and the reaction proceeded at 37 °C for 10 min. .. DNA was immediately mixed with loading dye and subjected to 0.5× TBE 4% (29:1) native PAGE at 100 V for 40 min.

    Article Title: Temporal Analysis of the Honey Bee Microbiome Reveals Four Novel Viruses and Seasonal Prevalence of Known Viruses, Nosema, and Crithidia
    Article Snippet: Light microscopy of live parasites was performed using a Leica DM6000 microscope equipped with Hamamatsu C4742-95 camera and Volocity Software (PerkinElmer). .. Bees from Crithidia positive hives were homogenized by TissueLyser as above and DNA extracted using the DNeasy kit for the initial PCR screens, after suspension in either PBS or 1× Micrococcal Nuclease Buffer (NEB).

    Negative Control:

    Article Title: DNA template strand sequencing of single-cells maps genomic rearrangements at high resolution
    Article Snippet: Plates were carefully removed from adaptors, and 90 μl cell-lysate supernatant was removed slowly and carefully using a long flexible gel-loading tip in order to avoid aspirating the nucleus. .. Next, 40 μl of 1.25× micrococcal nuclease (MNase) master mix (62.5 mM Tris-HCl pH 7.9, 6.25 mM CaCl, 0.03125 U/μl MNase enzyme, New England Biolabs) was added to each well containing a nucleus (as well as to no-cell negative-control wells containing only lysis buffer). .. Reactions were mixed 20–30 times using a pipettor and incubated at room temperature for 5 min.

    Binding Assay:

    Article Title: Characterization of a protein–protein interaction within the SigO–RsoA two-subunit σ factor: the σ70 region 2.3-like segment of RsoA mediates interaction with SigO
    Article Snippet: Cell pellets were subjected to two freeze–thaw cycles and the pellets were re-suspended in 200 µl of 10 mM Tris/HCl (pH 8.0), supplemented with 1× micrococcal nuclease buffer and 10 000 U micrococcal nuclease (NEB) and incubated at room temperature for 30 min until cells were lysed and genomic DNA was digested. .. Cell pellets were subjected to two freeze–thaw cycles and the pellets were re-suspended in 200 µl of 10 mM Tris/HCl (pH 8.0), supplemented with 1× micrococcal nuclease buffer and 10 000 U micrococcal nuclease (NEB) and incubated at room temperature for 30 min until cells were lysed and genomic DNA was digested.

    Article Title: Determinants of nucleosome positioning and their influence on plant gene expression
    Article Snippet: For isolating nucleosome gDNA, the nuclei were purified from one aliquot with nuclei extraction buffer (10 mM Hepes [pH 7.6], 5 mM potassium chloride, 5 mM magnesium chloride, 1 M sucrose, 14 mM β-mercaptoethanol, 0.6% Triton X-100, 0.4 mM phenylmethanesulfonyl fluoride, 1× ethylenediaminetetraacetic acid-free protease inhibitor [Roche]), digested with 0.4 units/μL micrococcal nuclease (MNase) (NEB) for 9 min, and purified with phenol/chloroform. .. To generate the naked gDNA sample, the gDNA was isolated without the nucleus isolation step as described previously , purified with phenol/chloroform to strip off proteins, and digested with 0.25 units/μL MNase (NEB) for 1.75 or 3 min.

    Article Title: Neutrophil accumulation and NET release contribute to thrombosis in HIT
    Article Snippet: To account for this effect when studying whether KKO binding to PF4-NET complexes increased resistance to DNase, we compared NET digestion by analyzing videos in which channels with and without KKO were included in the same visual field and exposed to UV light for the same amount of time. .. Similar studies were done using 100 U/ml bacterial-derived micrococcal nuclease (New England Biolabs).

    FACS:

    Article Title: DNA template strand sequencing of single-cells maps genomic rearrangements at high resolution
    Article Snippet: Paragraph title: FACS sorting and genomic DNA fragmentation ... Next, 40 μl of 1.25× micrococcal nuclease (MNase) master mix (62.5 mM Tris-HCl pH 7.9, 6.25 mM CaCl, 0.03125 U/μl MNase enzyme, New England Biolabs) was added to each well containing a nucleus (as well as to no-cell negative-control wells containing only lysis buffer).

    Homogenization:

    Article Title: CELF RNA binding proteins promote axon regeneration in C. elegans and mammals through alternative splicing of Syntaxins
    Article Snippet: Worms were lysed by sonication in Homogenization Buffer (100 mM NaCl, 25 mM HEPES, 250 μM EDTA, 2 mM DTT, 0.1% NP-40, 25 units/ml RNasin and Protease Inhibitors). .. Beads were incubated with 500 μl of Micrococcal Nuclease Reaction Buffer (50 mM Tris-Cl pH 7.9, 5 mM CaCl2 ) containing 1 ng of Micrococcal Nuclease (NEB) for a total of 10 min at 4°C with intermittent shaking on a Thermomixer (Eppendorf) (1200 rpm for 1 min and then 1200 rpm for 15 sec every 3 min).

    Article Title: Planarian MBD2/3 is required for adult stem cell pluripotency independently of DNA methylation
    Article Snippet: Worms were disrupted by homogenisation and DNA purified by two rounds of phenol-chloroform extraction and ethanol precipitation. .. Each 20 ml reaction contained S. mediterranea DNA, 5 units of Antarctic Phosphatase (NEB) and 2 mU Snake venom Phosphodiesterase (Phosphodiesterase I from Crotalus adamentus venom; Sigma) in a buffer that was 0.5×Micrococcal Nuclease Buffer (NEB) and 0.5×Antarctic Phosphatase Buffer (NEB).

    Ethanol Precipitation:

    Article Title: Planarian MBD2/3 is required for adult stem cell pluripotency independently of DNA methylation
    Article Snippet: Worms were disrupted by homogenisation and DNA purified by two rounds of phenol-chloroform extraction and ethanol precipitation. .. Each 20 ml reaction contained S. mediterranea DNA, 5 units of Antarctic Phosphatase (NEB) and 2 mU Snake venom Phosphodiesterase (Phosphodiesterase I from Crotalus adamentus venom; Sigma) in a buffer that was 0.5×Micrococcal Nuclease Buffer (NEB) and 0.5×Antarctic Phosphatase Buffer (NEB).

    FLAG-tag:

    Article Title: Characterization of a protein–protein interaction within the SigO–RsoA two-subunit σ factor: the σ70 region 2.3-like segment of RsoA mediates interaction with SigO
    Article Snippet: To directly detect interactions between SigO and RsoA, we fused the 5′ half (codons 1–105) of sigO to the T18 cyaA gene fragment in pUT18C and imparted a FLAG epitope 3′ to sigO . .. Cell pellets were subjected to two freeze–thaw cycles and the pellets were re-suspended in 200 µl of 10 mM Tris/HCl (pH 8.0), supplemented with 1× micrococcal nuclease buffer and 10 000 U micrococcal nuclease (NEB) and incubated at room temperature for 30 min until cells were lysed and genomic DNA was digested.

    Clear Native PAGE:

    Article Title: The ATP-dependent chromatin remodeling enzymes CHD6, CHD7, and CHD8 exhibit distinct nucleosome binding and remodeling activities
    Article Snippet: After 30 min, 1.5 mm final CaCl2 and 1 gel unit of MNase (New England Biolabs, M0247S) were supplemented, and the reaction proceeded at 37 °C for 10 min. .. After 30 min, 1.5 mm final CaCl2 and 1 gel unit of MNase (New England Biolabs, M0247S) were supplemented, and the reaction proceeded at 37 °C for 10 min.

    Liquid Chromatography with Mass Spectroscopy:

    Article Title: Planarian MBD2/3 is required for adult stem cell pluripotency independently of DNA methylation
    Article Snippet: Up to 5 mg of purified DNA was digested to nucleosides for subsequent LC–MS analysis. .. Each 20 ml reaction contained S. mediterranea DNA, 5 units of Antarctic Phosphatase (NEB) and 2 mU Snake venom Phosphodiesterase (Phosphodiesterase I from Crotalus adamentus venom; Sigma) in a buffer that was 0.5×Micrococcal Nuclease Buffer (NEB) and 0.5×Antarctic Phosphatase Buffer (NEB).

    Lysis:

    Article Title: ASSAY DEVELOPMENT AND HIGH THROUGHPUT SCREENING FOR INHIBITORS OF KAPOSI'S SARCOMA-ASSOCIATED HERPESVIRUS N-TERMINAL LANA BINDING TO NUCLEOSOMES
    Article Snippet: Flow-through containing RBC nuclei was centrifuged 5 min at 3500xg and pelleted nuclei were washed once with 10 volumes RBC lysis buffer followed by two additional washes (or until the supernatant and pellet were no longer red) with washing buffer (RBC lysis buffer without NP-40). .. Nuclei were then resuspended in micronuclease (MNase) buffer (15mM HEPES pH 7.5, 65mM NaCl, 65mM KCl, 2mM MgCl2 , 5mM CaCl2 and Complete EDTA-Free protease inhibitors cocktail tablet [Roche]) and incubated 2h at 37°C with 800 kunitz units MNase (NEB #M0247S)/10mL MNase buffer, to generate short chromatin of 1-6 nucleosomes long.

    Article Title: N6-methyladenine is an epigenetic marker of mammalian early life stress
    Article Snippet: Samples were washed 2x with PBS and cOmplete protease inhibitor (CPI) tablets (Roche, Basel, Switzerland; catalog #04693116001) then resuspended in 400 μL RIPA lysis buffer + CPI tablets. .. Chromatin shearing was obtained using 1000 gel units of Micrococcal Nuclease (MNase) (New England Biolabs; catalog #M0247S); samples were incubated with Proteinase K to ensure efficient shearing of DNA (between 300 bp–900 bp).

    Article Title: TRF2 is recruited to the pre-initiation complex as a testis-specific subunit of TFIIA/ALF to promote haploid cell gene expression
    Article Snippet: Nuclei were washed once with 1X PBS and spin and resuspended in 2 volumes of MNase lysis buffer (50 mM Tris pH8, 0.5% Na deoxycholate). .. The lysate was sonicated for 30 sec as described above, CaCl2 and MgCl2 were added to 5 mM final concentration each and digestion was performed with MNase (10 gel units/uL final) (M0247S, New England BioLabs) for 10 min of incubation.

    Article Title: Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor
    Article Snippet: SBP-tagged eIF4A was purified as described in “RIP-Seq”, without DMSO or RocA treatment. .. The beads tethering SBP-eIF4A were treated with 1x Micrococcal Nuclease Buffer (NEB), 0.5x lysis buffer, 0.5% Triton X-100, and 200 U/μl Micrococcal Nuclease (NEB) in 30 μl at 25 °C for 30 min, washed 5 times with lysis buffer containing 1% Triton X-100, 1M NaCl, and 5 mM EGTA pH 7.4, and rinsed twice with lysis buffer containing 0.1% Triton X-100. .. The beads were incubated in lysis buffer containing 0.1% Triton X-100, 2 mM AMP-PNP, 0.33 U/μl SUPERase In RNase Inhibitor (Invitrogen), 1 μM N30 RNA [(N)30 CTGTAGGCACCATCAAT , bold characters represent DNA sequence for reverse transcription primer hybridization] in 30 μl at 37 °C for 30 min, and washed 5 times with lysis buffer containing 0.1% Triton X-100, 2 mM AMP-PNP, and 0.1% DMSO.

    Article Title: DNA template strand sequencing of single-cells maps genomic rearrangements at high resolution
    Article Snippet: Plates were carefully removed from adaptors, and 90 μl cell-lysate supernatant was removed slowly and carefully using a long flexible gel-loading tip in order to avoid aspirating the nucleus. .. Next, 40 μl of 1.25× micrococcal nuclease (MNase) master mix (62.5 mM Tris-HCl pH 7.9, 6.25 mM CaCl, 0.03125 U/μl MNase enzyme, New England Biolabs) was added to each well containing a nucleus (as well as to no-cell negative-control wells containing only lysis buffer). .. Reactions were mixed 20–30 times using a pipettor and incubated at room temperature for 5 min.

    Article Title: Transient Downregulation of Nanog and Oct4 Induced by DETA/NO Exposure in Mouse Embryonic Stem Cells Leads to Mesodermal/Endodermal Lineage Differentiation
    Article Snippet: Cells were then resuspended in lysis buffer containing 10 mM Tris HCl (pH 8.0), 10 mM NaCl, 3 mM MgCl2 , 0.5 mM DTT, and protease inhibitors for 10 min on ice. .. Pellets were then resuspended in washing buffer supplemented with 3 mM CaCl2 , protease inhibitors, 0.5 mM DTT, and 5–10 μ L of 1 : 200 micrococcal nuclease (New England BioLabs) and incubated for 20 min at 37°C with orbital shaking.

    Article Title: Androgen Receptor Serine 81 Phosphorylation Mediates Chromatin Binding and Transcriptional Activation
    Article Snippet: Cellular fractionation analysis using the NE-PER kit was carried out as described by the manufacturer (catalog no. 78835, Pierce), and using the Triton lysis buffer (TLB) ( ). .. The insoluble fraction (pellet left from cytoplasmic and nuclear protein extraction) was suspended in 40 μl of micrococcal nuclease digestion buffer (M0247, New England Biolabs), kept at room temperature, and treated with 600 units of micrococcal nuclease for 2 min.

    Cross-linking Immunoprecipitation:

    Article Title: CELF RNA binding proteins promote axon regeneration in C. elegans and mammals through alternative splicing of Syntaxins
    Article Snippet: Paragraph title: Crosslinking Immunoprecipitation and deep sequencing (CLIP-seq) in C. elegans ... Beads were incubated with 500 μl of Micrococcal Nuclease Reaction Buffer (50 mM Tris-Cl pH 7.9, 5 mM CaCl2 ) containing 1 ng of Micrococcal Nuclease (NEB) for a total of 10 min at 4°C with intermittent shaking on a Thermomixer (Eppendorf) (1200 rpm for 1 min and then 1200 rpm for 15 sec every 3 min).

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    New England Biolabs mnase
    A possible model of low-level nucleoid organization. a A fragment of EM image of E. coli nucleoid (adapted from [ 21 ]). b Genomic <t>DNA</t> (gray) forms loops with A-tract clusters (cyan) located at apexes and <t>MNase</t> resistant fragments (black) occupying loop ‘stems’. c A blow-up of a DNA loop, containing several A-tracts (cyan) and two MNase resistant fragments (black)
    Mnase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs mnase seq samples
    Histone deacetylation contributes to stable gene silencing. ( A ) RT-PCR showed that 1 ng/ml TSA for 24 hr significantly relieved Ikaros-induced reduced repression of Igll and Myc primary transcripts. Mean ± SE, 3 independent biological replicates. ( B ) <t>MNase</t> PCR showed that 1 ng/ml TSA for 24 hr did not significantly affect protection of 80–120 bp amplicons (short, left) but significantly reduced protection of 130–140 bp amplicons (long, right) at the Igll1 promoter. Mean ± SE, 3 independent biological replicates. ( C ) ChIP-PCR to assess Ikaros-induced recruitment of histone H2B to the Igll1 promoter between control cells and cells treated with 1 ng/ml TSA for 24 hr. Enrichment was normalised to total H3. Mean ± SE, 3 independent biological replicates. TSA significantly blunted the Ikaros-induced increase the H2B/H3 ratio at the Igll1 promoter and TSS. ( D ) 3D DNA-FISH to monitor the position of Igll1 alleles (green) relative to γ-satellite DNA (red, blue is DAPI). The percentage of Igll1 alleles associated with γ-satellite DNA is shown as mean ± SE. Where indicated, cells were treated over night with TSA (1 ng/ml) and/or <t>4-OHT.</t> At least 300 Igll1 alleles were scored for each experimental condition across 3 independent biological replicates. The impact of TSA was statistically significant across replicates (p=9.54 × 10-18 GLM binomial logit). DOI: http://dx.doi.org/10.7554/eLife.22767.021 10.7554/eLife.22767.022 Numerical data used to generate Figure 6A,B,C and D . DOI: http://dx.doi.org/10.7554/eLife.22767.022
    Mnase Seq Samples, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    A possible model of low-level nucleoid organization. a A fragment of EM image of E. coli nucleoid (adapted from [ 21 ]). b Genomic DNA (gray) forms loops with A-tract clusters (cyan) located at apexes and MNase resistant fragments (black) occupying loop ‘stems’. c A blow-up of a DNA loop, containing several A-tracts (cyan) and two MNase resistant fragments (black)

    Journal: BMC Microbiology

    Article Title: Organization of DNA in a bacterial nucleoid

    doi: 10.1186/s12866-016-0637-3

    Figure Lengend Snippet: A possible model of low-level nucleoid organization. a A fragment of EM image of E. coli nucleoid (adapted from [ 21 ]). b Genomic DNA (gray) forms loops with A-tract clusters (cyan) located at apexes and MNase resistant fragments (black) occupying loop ‘stems’. c A blow-up of a DNA loop, containing several A-tracts (cyan) and two MNase resistant fragments (black)

    Article Snippet: The wild type genomic DNA was purified using Wizard Genomic DNA Purification Kit (Promega) and treated with MNase (New England Biolabs) at 12 °C.

    Techniques: Electron Microscopy

    Analysis of the DNA fragments from in vivo MNase digestion of nucleoid in wild type E. coli ( a ) and in vitro MNase digestion of purified genomic DNA ( b ) in agarose gel. a Wild type cells with the empty vector (lanes 2, 3) and MNase-expressing vector (lanes 4-8) were supplemented with arabinose (lane 6), CaCl 2 (lane 5) or both (lanes 3, 7, 8). Digestion reactions were stopped 1 minute (lane 7) or 5 minutes (lanes 3, 5, 8) after CaCl 2 was added. Lanes 1 and 9 show DNA molecular weight marker. b Lane 1, purified wild type genomic DNA; lanes 2-6, a time course of in vitro MNase digestion of the wild type genomic DNA; lane 7, DNA molecular marker

    Journal: BMC Microbiology

    Article Title: Organization of DNA in a bacterial nucleoid

    doi: 10.1186/s12866-016-0637-3

    Figure Lengend Snippet: Analysis of the DNA fragments from in vivo MNase digestion of nucleoid in wild type E. coli ( a ) and in vitro MNase digestion of purified genomic DNA ( b ) in agarose gel. a Wild type cells with the empty vector (lanes 2, 3) and MNase-expressing vector (lanes 4-8) were supplemented with arabinose (lane 6), CaCl 2 (lane 5) or both (lanes 3, 7, 8). Digestion reactions were stopped 1 minute (lane 7) or 5 minutes (lanes 3, 5, 8) after CaCl 2 was added. Lanes 1 and 9 show DNA molecular weight marker. b Lane 1, purified wild type genomic DNA; lanes 2-6, a time course of in vitro MNase digestion of the wild type genomic DNA; lane 7, DNA molecular marker

    Article Snippet: The wild type genomic DNA was purified using Wizard Genomic DNA Purification Kit (Promega) and treated with MNase (New England Biolabs) at 12 °C.

    Techniques: In Vivo, In Vitro, Purification, Agarose Gel Electrophoresis, Plasmid Preparation, Expressing, Molecular Weight, Marker

    Genome-wide distribution of sequenced tags. a Tag frequencies for the entire E. coli genome. Frequencies of tags mapped on the positive and negative strands are shown with red and blue bars respectively. a , c Schematic illustrations of tag cross-correlation ( b ) and auto-correlation analyses ( c ). MNase resistant fragments are shown with grey rectangles. Vertical red and blue arrows represent 5’-ends of the digestion fragments mapping to the DNA positive and negative strands respectively

    Journal: BMC Microbiology

    Article Title: Organization of DNA in a bacterial nucleoid

    doi: 10.1186/s12866-016-0637-3

    Figure Lengend Snippet: Genome-wide distribution of sequenced tags. a Tag frequencies for the entire E. coli genome. Frequencies of tags mapped on the positive and negative strands are shown with red and blue bars respectively. a , c Schematic illustrations of tag cross-correlation ( b ) and auto-correlation analyses ( c ). MNase resistant fragments are shown with grey rectangles. Vertical red and blue arrows represent 5’-ends of the digestion fragments mapping to the DNA positive and negative strands respectively

    Article Snippet: The wild type genomic DNA was purified using Wizard Genomic DNA Purification Kit (Promega) and treated with MNase (New England Biolabs) at 12 °C.

    Techniques: Genome Wide

    Ectopically expressed histone H3p localizes to the nucleus during parasite asexual development and incorporates into nucleosomes Indirect immunofluorescence assays were performed to determine the localization of ectopically expressed PfH3p‐HA in ring (R), trophozoite (T), and schizont (S) stages of Plasmodium falciparum asexual growth. PfH3p‐HA was detected using anti‐HA antibodies (green) and endogenous histone H3 with anti‐histone H3 N‐terminal antibodies (red). DAPI (blue) was used to stain the nucleus. Scale bar = 5 μm. Nuclei isolated from wild‐type (WT) or PfH3p‐HA‐expressing (WT + PfH3p‐HA) schizont‐stage parasites were treated with 4 U/ml of micrococcal nuclease (MNase) for the indicated amounts of time, the DNA purified and migrated on a 2% agarose gel, and stained with ethidium bromide. Mononucleosomes purified after 10 min of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis and either stained with Coomassie Brilliant Blue (C.B.) or visualized by immunoblotting with anti‐HA (α‐HA) or anti‐C‐terminal histone H3 (α‐H3c) antibodies. Co‐immunoprecipitation (IP) experiments of purified mononucleosomes obtained from wild‐type (WT) or transfected (WT + PfH3p‐HA) schizont‐stage parasites were performed with either anti‐HA antibodies or mouse IgG. Immunoprecipitated products (right panel) were analyzed by immunoblotting using anti‐HA or anti‐histone H4 antibodies. Source data are available online for this figure.

    Journal: EMBO Reports

    Article Title: Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum

    doi: 10.15252/embr.201846331

    Figure Lengend Snippet: Ectopically expressed histone H3p localizes to the nucleus during parasite asexual development and incorporates into nucleosomes Indirect immunofluorescence assays were performed to determine the localization of ectopically expressed PfH3p‐HA in ring (R), trophozoite (T), and schizont (S) stages of Plasmodium falciparum asexual growth. PfH3p‐HA was detected using anti‐HA antibodies (green) and endogenous histone H3 with anti‐histone H3 N‐terminal antibodies (red). DAPI (blue) was used to stain the nucleus. Scale bar = 5 μm. Nuclei isolated from wild‐type (WT) or PfH3p‐HA‐expressing (WT + PfH3p‐HA) schizont‐stage parasites were treated with 4 U/ml of micrococcal nuclease (MNase) for the indicated amounts of time, the DNA purified and migrated on a 2% agarose gel, and stained with ethidium bromide. Mononucleosomes purified after 10 min of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis and either stained with Coomassie Brilliant Blue (C.B.) or visualized by immunoblotting with anti‐HA (α‐HA) or anti‐C‐terminal histone H3 (α‐H3c) antibodies. Co‐immunoprecipitation (IP) experiments of purified mononucleosomes obtained from wild‐type (WT) or transfected (WT + PfH3p‐HA) schizont‐stage parasites were performed with either anti‐HA antibodies or mouse IgG. Immunoprecipitated products (right panel) were analyzed by immunoblotting using anti‐HA or anti‐histone H4 antibodies. Source data are available online for this figure.

    Article Snippet: Mononucleosomes were prepared from freshly isolated nuclei by digestion with 4 U MNase (NEB) for different amounts of time at 33°C; digestion was stopped by adding 10 mM EGTA pH 8.0.

    Techniques: Immunofluorescence, Staining, Isolation, Expressing, Purification, Agarose Gel Electrophoresis, Polyacrylamide Gel Electrophoresis, Immunoprecipitation, Transfection

    Isolation of cross-linked ORF57-RNA complexes for HITS-CLIP analysis. Top Nitrocellulose membrane from an ORF57 immunoprecipitation performed under HITS-CLIP conditions. Cross-linked RNA fragments were end-labeled with 32 P to be visualized by Phosphoimager. Cells were induced to undergo lytic reactivation, exposed to UV and/or treated with high or low concentrations of MNase as indicated. The samples lacking an ORF57 antibody (lane 1) were precipitated with pre-bleed antibodies from the same rabbit. Lanes 6 and 7 are a dark exposure of lanes 4 and 5. The dashed white box indicates the position of the ORF57 complex cut from the membrane for library preparation. Bottom Western blot of the same HITS-CLIP samples shown in the top panel. Affinity purified rabbit anti-ORF57 was used to detect ORF57. Positions of molecular weight markers are shown on the left. On the right, a single asterisk marks the position of a contaminating ~37 kDa protein; double and triple asterisks mark positions of putative ORF57 homodimers and homotrimers bound to the same RNA. The doublet is likely due to an ORF57 cleavage product [ 104 ].

    Journal: PLoS Pathogens

    Article Title: HITS-CLIP Analysis Uncovers a Link between the Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein and Host Pre-mRNA Metabolism

    doi: 10.1371/journal.ppat.1004652

    Figure Lengend Snippet: Isolation of cross-linked ORF57-RNA complexes for HITS-CLIP analysis. Top Nitrocellulose membrane from an ORF57 immunoprecipitation performed under HITS-CLIP conditions. Cross-linked RNA fragments were end-labeled with 32 P to be visualized by Phosphoimager. Cells were induced to undergo lytic reactivation, exposed to UV and/or treated with high or low concentrations of MNase as indicated. The samples lacking an ORF57 antibody (lane 1) were precipitated with pre-bleed antibodies from the same rabbit. Lanes 6 and 7 are a dark exposure of lanes 4 and 5. The dashed white box indicates the position of the ORF57 complex cut from the membrane for library preparation. Bottom Western blot of the same HITS-CLIP samples shown in the top panel. Affinity purified rabbit anti-ORF57 was used to detect ORF57. Positions of molecular weight markers are shown on the left. On the right, a single asterisk marks the position of a contaminating ~37 kDa protein; double and triple asterisks mark positions of putative ORF57 homodimers and homotrimers bound to the same RNA. The doublet is likely due to an ORF57 cleavage product [ 104 ].

    Article Snippet: For RNA digestion, MNase (New England Biolabs) was diluted to 10 gel units/μL in RIPA buffer and 5 μL of this freshly diluted 1:200 stock was added to the extract.

    Techniques: Isolation, Cross-linking Immunoprecipitation, Immunoprecipitation, Labeling, Western Blot, Affinity Purification, Molecular Weight

    Histone deacetylation contributes to stable gene silencing. ( A ) RT-PCR showed that 1 ng/ml TSA for 24 hr significantly relieved Ikaros-induced reduced repression of Igll and Myc primary transcripts. Mean ± SE, 3 independent biological replicates. ( B ) MNase PCR showed that 1 ng/ml TSA for 24 hr did not significantly affect protection of 80–120 bp amplicons (short, left) but significantly reduced protection of 130–140 bp amplicons (long, right) at the Igll1 promoter. Mean ± SE, 3 independent biological replicates. ( C ) ChIP-PCR to assess Ikaros-induced recruitment of histone H2B to the Igll1 promoter between control cells and cells treated with 1 ng/ml TSA for 24 hr. Enrichment was normalised to total H3. Mean ± SE, 3 independent biological replicates. TSA significantly blunted the Ikaros-induced increase the H2B/H3 ratio at the Igll1 promoter and TSS. ( D ) 3D DNA-FISH to monitor the position of Igll1 alleles (green) relative to γ-satellite DNA (red, blue is DAPI). The percentage of Igll1 alleles associated with γ-satellite DNA is shown as mean ± SE. Where indicated, cells were treated over night with TSA (1 ng/ml) and/or 4-OHT. At least 300 Igll1 alleles were scored for each experimental condition across 3 independent biological replicates. The impact of TSA was statistically significant across replicates (p=9.54 × 10-18 GLM binomial logit). DOI: http://dx.doi.org/10.7554/eLife.22767.021 10.7554/eLife.22767.022 Numerical data used to generate Figure 6A,B,C and D . DOI: http://dx.doi.org/10.7554/eLife.22767.022

    Journal: eLife

    Article Title: A high-resolution map of transcriptional repression

    doi: 10.7554/eLife.22767

    Figure Lengend Snippet: Histone deacetylation contributes to stable gene silencing. ( A ) RT-PCR showed that 1 ng/ml TSA for 24 hr significantly relieved Ikaros-induced reduced repression of Igll and Myc primary transcripts. Mean ± SE, 3 independent biological replicates. ( B ) MNase PCR showed that 1 ng/ml TSA for 24 hr did not significantly affect protection of 80–120 bp amplicons (short, left) but significantly reduced protection of 130–140 bp amplicons (long, right) at the Igll1 promoter. Mean ± SE, 3 independent biological replicates. ( C ) ChIP-PCR to assess Ikaros-induced recruitment of histone H2B to the Igll1 promoter between control cells and cells treated with 1 ng/ml TSA for 24 hr. Enrichment was normalised to total H3. Mean ± SE, 3 independent biological replicates. TSA significantly blunted the Ikaros-induced increase the H2B/H3 ratio at the Igll1 promoter and TSS. ( D ) 3D DNA-FISH to monitor the position of Igll1 alleles (green) relative to γ-satellite DNA (red, blue is DAPI). The percentage of Igll1 alleles associated with γ-satellite DNA is shown as mean ± SE. Where indicated, cells were treated over night with TSA (1 ng/ml) and/or 4-OHT. At least 300 Igll1 alleles were scored for each experimental condition across 3 independent biological replicates. The impact of TSA was statistically significant across replicates (p=9.54 × 10-18 GLM binomial logit). DOI: http://dx.doi.org/10.7554/eLife.22767.021 10.7554/eLife.22767.022 Numerical data used to generate Figure 6A,B,C and D . DOI: http://dx.doi.org/10.7554/eLife.22767.022

    Article Snippet: For MNase-seq, 50 ng DNA after MNase digestion (carrier ethanol treatment for 6 hr or 0.5 μM 4-OHT treatment for 6 hr) were used to prepare MNase-seq samples (Next Ultra, NEB) without size selection steps.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Fluorescence In Situ Hybridization

    Ikaros controls promoter accessibility through NuRD-associated chromatin remodeling. ( A ) Left: CHD4 expression in control and Chd4 shRNA cells by western blotting. Tubulin is a loading control. One of 5 independent biological replicates. Right: Experimental outline. ( B ) MNase-PCR at the Igll1 and Myc promoters in control (black) or Chd4 shRNA cells (red) at the indicated times after 4-OHT. Mean ± SE, 3 independent biological replicates. Chd4 shRNA significantly reduced the Ikaros-induced increase in nucleosome occupancy at 15, 30 and 120 min at the Igll1 promoter and at 30 and 120 min at the Myc promoter. ( C ) RNAP2 ChIP-PCR (top) and MNase-PCR (bottom) at the Igll1 and Myc promoters after 4-OHT in control (black) or Chd4 shRNA cells (red). Mean ± SE, 3 independent biological replicates. RNAP2 binding was significantly reduced in control cells but not in Chd4 shRNA-treated cells from 5 to 120 min after 4-OHT at the Igll1 and the Myc promoter. Primary transcripts were significantly reduced in control but not in Chd4 shRNA-treated cells at 15 and 30 min for Igll1 and at 30 and 120 min for Myc . ( D ) ChIP-PCR for CHD4 (black), MBD3 (grey) and BRG1 (orange) at the Igll1 promoters at the indicated times after 4-OHT. Mean ± SE, 5 independent biological replicates for CHD4 and BRG1, 3 independent biological replicates for MBD3. CHD4 and MBD3 binding at the Igll1 promoter were significantly increased from 5 to 60 min. BRG1 binding was significantly decreased from 30 to 120 min. DOI: http://dx.doi.org/10.7554/eLife.22767.015 10.7554/eLife.22767.016 Numerical data used to generate Figure 4B,C and D . DOI: http://dx.doi.org/10.7554/eLife.22767.016

    Journal: eLife

    Article Title: A high-resolution map of transcriptional repression

    doi: 10.7554/eLife.22767

    Figure Lengend Snippet: Ikaros controls promoter accessibility through NuRD-associated chromatin remodeling. ( A ) Left: CHD4 expression in control and Chd4 shRNA cells by western blotting. Tubulin is a loading control. One of 5 independent biological replicates. Right: Experimental outline. ( B ) MNase-PCR at the Igll1 and Myc promoters in control (black) or Chd4 shRNA cells (red) at the indicated times after 4-OHT. Mean ± SE, 3 independent biological replicates. Chd4 shRNA significantly reduced the Ikaros-induced increase in nucleosome occupancy at 15, 30 and 120 min at the Igll1 promoter and at 30 and 120 min at the Myc promoter. ( C ) RNAP2 ChIP-PCR (top) and MNase-PCR (bottom) at the Igll1 and Myc promoters after 4-OHT in control (black) or Chd4 shRNA cells (red). Mean ± SE, 3 independent biological replicates. RNAP2 binding was significantly reduced in control cells but not in Chd4 shRNA-treated cells from 5 to 120 min after 4-OHT at the Igll1 and the Myc promoter. Primary transcripts were significantly reduced in control but not in Chd4 shRNA-treated cells at 15 and 30 min for Igll1 and at 30 and 120 min for Myc . ( D ) ChIP-PCR for CHD4 (black), MBD3 (grey) and BRG1 (orange) at the Igll1 promoters at the indicated times after 4-OHT. Mean ± SE, 5 independent biological replicates for CHD4 and BRG1, 3 independent biological replicates for MBD3. CHD4 and MBD3 binding at the Igll1 promoter were significantly increased from 5 to 60 min. BRG1 binding was significantly decreased from 30 to 120 min. DOI: http://dx.doi.org/10.7554/eLife.22767.015 10.7554/eLife.22767.016 Numerical data used to generate Figure 4B,C and D . DOI: http://dx.doi.org/10.7554/eLife.22767.016

    Article Snippet: For MNase-seq, 50 ng DNA after MNase digestion (carrier ethanol treatment for 6 hr or 0.5 μM 4-OHT treatment for 6 hr) were used to prepare MNase-seq samples (Next Ultra, NEB) without size selection steps.

    Techniques: Expressing, shRNA, Western Blot, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Binding Assay

    Nuclear translocation of Ikaros by proteolytic cleavage of Ikaros fusion proteins. ( A ) Schematic representation of Ikaros translocation induced by the proteolytic cleavage of Ikaros-TEV-ERt2 by Tobacco Etch Virus (TEV) protease. Ikaros-TEV-ERt2 fusion proteins are retained in the cytompasm until cleavage. The separate N- and C-termini of TEV protease are fused to FKBP and FRB, respectively. TEV activity is restored by the addition of rapamycin (R) ( Wehr et al., 2006 ). Cleavage separates Ikaros from ERt2. The efficiency of inducible cleavage after rapamycin (2 hr, 25 nM) is monitored by western blotting using Ikaros antibodies (right). Actin was used as loading control. Representative of 3 independent biological replicates. ( B ) MNase digestion followed by PCR was used to determine changes in the accessibility of the Igll1 and Myc promoters after triptolide-induced removal of RNAP2. The experiment complements the data shown in Figure 3B and C , but Split-TEV was used instead of 4-OHT-induced nuclear translocation of Ikaros. Split-TEV was activated by the addition of rapamycin at 25 nM for 2 hr. Mean ± SE, 3 independent biological replicates. Ikaros induction and tripolide-mediated RNAP2 depletion may synergise in increasing the nucleosome occupancy of the Igll1 promoter. DOI: http://dx.doi.org/10.7554/eLife.22767.013 10.7554/eLife.22767.014 Numerical data used to generate Figure 3—figure supplement 1A,B,C and D . DOI: http://dx.doi.org/10.7554/eLife.22767.014

    Journal: eLife

    Article Title: A high-resolution map of transcriptional repression

    doi: 10.7554/eLife.22767

    Figure Lengend Snippet: Nuclear translocation of Ikaros by proteolytic cleavage of Ikaros fusion proteins. ( A ) Schematic representation of Ikaros translocation induced by the proteolytic cleavage of Ikaros-TEV-ERt2 by Tobacco Etch Virus (TEV) protease. Ikaros-TEV-ERt2 fusion proteins are retained in the cytompasm until cleavage. The separate N- and C-termini of TEV protease are fused to FKBP and FRB, respectively. TEV activity is restored by the addition of rapamycin (R) ( Wehr et al., 2006 ). Cleavage separates Ikaros from ERt2. The efficiency of inducible cleavage after rapamycin (2 hr, 25 nM) is monitored by western blotting using Ikaros antibodies (right). Actin was used as loading control. Representative of 3 independent biological replicates. ( B ) MNase digestion followed by PCR was used to determine changes in the accessibility of the Igll1 and Myc promoters after triptolide-induced removal of RNAP2. The experiment complements the data shown in Figure 3B and C , but Split-TEV was used instead of 4-OHT-induced nuclear translocation of Ikaros. Split-TEV was activated by the addition of rapamycin at 25 nM for 2 hr. Mean ± SE, 3 independent biological replicates. Ikaros induction and tripolide-mediated RNAP2 depletion may synergise in increasing the nucleosome occupancy of the Igll1 promoter. DOI: http://dx.doi.org/10.7554/eLife.22767.013 10.7554/eLife.22767.014 Numerical data used to generate Figure 3—figure supplement 1A,B,C and D . DOI: http://dx.doi.org/10.7554/eLife.22767.014

    Article Snippet: For MNase-seq, 50 ng DNA after MNase digestion (carrier ethanol treatment for 6 hr or 0.5 μM 4-OHT treatment for 6 hr) were used to prepare MNase-seq samples (Next Ultra, NEB) without size selection steps.

    Techniques: Translocation Assay, Activity Assay, Western Blot, Polymerase Chain Reaction

    Interdependence of silencing mechanisms leveraged by Ikaros. ( A ) 3D DNA-FISH to monitor the position of Igll1 alleles (green) relative to γ-satellite DNA (red, blue is DAPI). The percentage of Igll1 alleles associated with γ-satellite DNA is shown as mean ± SE. Where indicated, control or sh Chd4 cells were treated over night with 4-OHT. At least 300 Igll1 alleles were scored for each experimental condition across 3 independent biological replicates. The impact of Chd4 knockdown was statistically significant across replicates (p=5.54×10-38 GLM binomial logit). ( B ) ChIP kinetics of Ikaros and EBF binding to the Igll1 promoter in control (left) and sh Chd4 cells (right). Increased binding of Ikaros to the Igll1 promoter was significant for both control and sh Chd4 cells, decreased binding of EBF1 was significant in control, but not in sh Chd4 cells. Mean ± SE, 3 independent biological replicates. Ikaros and EBF1 binding at 15, 30 and 120 min were significantly higher in sh Chd4 than control cells. ( C ) MNase-seq data from 3 independent biological replicates were integrated with Ikaros ChIP-seq data to show nucleosome occupancy at Ikaros binding peaks before and 6 hr after nuclear translocation of Ikaros. ( D ) Dynamics of Ikaros binding, RNAP2 eviction, loss of primary transcripts, nucleosome invasion, and histone deacetylation. DOI: http://dx.doi.org/10.7554/eLife.22767.023 10.7554/eLife.22767.024 Numerical data used to generate Figure 7A and B . DOI: http://dx.doi.org/10.7554/eLife.22767.024

    Journal: eLife

    Article Title: A high-resolution map of transcriptional repression

    doi: 10.7554/eLife.22767

    Figure Lengend Snippet: Interdependence of silencing mechanisms leveraged by Ikaros. ( A ) 3D DNA-FISH to monitor the position of Igll1 alleles (green) relative to γ-satellite DNA (red, blue is DAPI). The percentage of Igll1 alleles associated with γ-satellite DNA is shown as mean ± SE. Where indicated, control or sh Chd4 cells were treated over night with 4-OHT. At least 300 Igll1 alleles were scored for each experimental condition across 3 independent biological replicates. The impact of Chd4 knockdown was statistically significant across replicates (p=5.54×10-38 GLM binomial logit). ( B ) ChIP kinetics of Ikaros and EBF binding to the Igll1 promoter in control (left) and sh Chd4 cells (right). Increased binding of Ikaros to the Igll1 promoter was significant for both control and sh Chd4 cells, decreased binding of EBF1 was significant in control, but not in sh Chd4 cells. Mean ± SE, 3 independent biological replicates. Ikaros and EBF1 binding at 15, 30 and 120 min were significantly higher in sh Chd4 than control cells. ( C ) MNase-seq data from 3 independent biological replicates were integrated with Ikaros ChIP-seq data to show nucleosome occupancy at Ikaros binding peaks before and 6 hr after nuclear translocation of Ikaros. ( D ) Dynamics of Ikaros binding, RNAP2 eviction, loss of primary transcripts, nucleosome invasion, and histone deacetylation. DOI: http://dx.doi.org/10.7554/eLife.22767.023 10.7554/eLife.22767.024 Numerical data used to generate Figure 7A and B . DOI: http://dx.doi.org/10.7554/eLife.22767.024

    Article Snippet: For MNase-seq, 50 ng DNA after MNase digestion (carrier ethanol treatment for 6 hr or 0.5 μM 4-OHT treatment for 6 hr) were used to prepare MNase-seq samples (Next Ultra, NEB) without size selection steps.

    Techniques: Fluorescence In Situ Hybridization, Chromatin Immunoprecipitation, Binding Assay, Translocation Assay