puc19  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94
    Name:
    Micrococcal Nuclease
    Description:
    Micrococcal Nuclease 320 000 gel units
    Catalog Number:
    m0247s
    Price:
    75
    Size:
    320 000 gel units
    Category:
    Exonucleases
    Buy from Supplier


    Structured Review

    New England Biolabs puc19
    Micrococcal Nuclease
    Micrococcal Nuclease 320 000 gel units
    https://www.bioz.com/result/puc19/product/New England Biolabs
    Average 94 stars, based on 612 article reviews
    Price from $9.99 to $1999.99
    puc19 - by Bioz Stars, 2020-07
    94/100 stars

    Images

    1) Product Images from "Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer"

    Article Title: Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00787-16

    In vitro analysis of RpnA endonuclease activity. (A) WT RpnA cleaves pUC19, RpnA-D63A does not cleave pUC19, and RpnA-D165A is more active on pUC19. The pUC19 DNA (29 nM, 50 μg/ml) is initially supercoiled but can be relaxed by nicks, linearized by double-strand cleavage, or cleaved further. The supercoiled (control), relaxed (Nb.BtsI), and linear (HindIII) forms are indicated. pUC19 was treated with RpnA-inactive RpnA-D63A or hyperactive RpnA-D165A (15 μM, 45 min). (B) Time course of an RpnA (7.5 μM)-pUC19 (29 nM) digest. Band intensity was compared to determine the relative amounts of supercoiled, nicked, and linear pUC19 at each time point. Over 90% of the supercoiled pUC19 was digested within 180 min. (C) RpnA endonuclease activity depends on divalent cation and is stimulated by Ca 2+ . The reaction buffer was 50 mM NaCl and 10 mM Tris, pH 8.0; the indicated additives were at 10 mM each. RpnA at 3.8 μM was added for 18 h. (D) RpnA cleavage products provide a DNA polymerase primer. pUC19 was digested with RpnA, DNase I, or micrococcal nuclease (MNase) to produce similar smears and then incubated with fluorescein-labeled dNTPs and the Klenow fragment of DNA polymerase. DNA was visualized by ethidium bromide (EtBr; left) or fluorescein (middle), with the two signals being merged at the right. RpnA- and DNase I-digested DNAs were effectively labeled, but micrococcal nuclease-digested DNA was not.
    Figure Legend Snippet: In vitro analysis of RpnA endonuclease activity. (A) WT RpnA cleaves pUC19, RpnA-D63A does not cleave pUC19, and RpnA-D165A is more active on pUC19. The pUC19 DNA (29 nM, 50 μg/ml) is initially supercoiled but can be relaxed by nicks, linearized by double-strand cleavage, or cleaved further. The supercoiled (control), relaxed (Nb.BtsI), and linear (HindIII) forms are indicated. pUC19 was treated with RpnA-inactive RpnA-D63A or hyperactive RpnA-D165A (15 μM, 45 min). (B) Time course of an RpnA (7.5 μM)-pUC19 (29 nM) digest. Band intensity was compared to determine the relative amounts of supercoiled, nicked, and linear pUC19 at each time point. Over 90% of the supercoiled pUC19 was digested within 180 min. (C) RpnA endonuclease activity depends on divalent cation and is stimulated by Ca 2+ . The reaction buffer was 50 mM NaCl and 10 mM Tris, pH 8.0; the indicated additives were at 10 mM each. RpnA at 3.8 μM was added for 18 h. (D) RpnA cleavage products provide a DNA polymerase primer. pUC19 was digested with RpnA, DNase I, or micrococcal nuclease (MNase) to produce similar smears and then incubated with fluorescein-labeled dNTPs and the Klenow fragment of DNA polymerase. DNA was visualized by ethidium bromide (EtBr; left) or fluorescein (middle), with the two signals being merged at the right. RpnA- and DNase I-digested DNAs were effectively labeled, but micrococcal nuclease-digested DNA was not.

    Techniques Used: In Vitro, Activity Assay, Incubation, Labeling

    2) 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

    Assembly of H2B K34ub and unmodified nucleosomes. ( A ) Left panel: SDS-PAGE of H2B K34ub- (lane 1) and unmodified (lane 2) histone octamers. Right panel: native PAGE of modified nucleosomes assembled on 147 bp 601 DNA using increasing amounts histones. ( B ) SDS-PAGE of faster- (lane 1) and slower- (lane 2) migrating H2B K34ub assembly products extracted from the native gel in A. The densitometric tracing of the gel lanes is shown on the right. Quantification, by ImageJ, is normalized to that of the histone H4, which is arbitrarily set as 1. ( C ) Native PAGE for samples prepared by serial dilution of 2M NaCl mixtures of 147 bp 601 DNA and modified or unmodified histones to 1 M NaCl. ( D ) Left panel: SDS-PAGE of recombinant linker histone H1 0 . Right panel: assembly of H2B K34ub modified and unmodified nucleosomes with increasing concentration of recombinant histone H1 0 .
    Figure Legend Snippet: Assembly of H2B K34ub and unmodified nucleosomes. ( A ) Left panel: SDS-PAGE of H2B K34ub- (lane 1) and unmodified (lane 2) histone octamers. Right panel: native PAGE of modified nucleosomes assembled on 147 bp 601 DNA using increasing amounts histones. ( B ) SDS-PAGE of faster- (lane 1) and slower- (lane 2) migrating H2B K34ub assembly products extracted from the native gel in A. The densitometric tracing of the gel lanes is shown on the right. Quantification, by ImageJ, is normalized to that of the histone H4, which is arbitrarily set as 1. ( C ) Native PAGE for samples prepared by serial dilution of 2M NaCl mixtures of 147 bp 601 DNA and modified or unmodified histones to 1 M NaCl. ( D ) Left panel: SDS-PAGE of recombinant linker histone H1 0 . Right panel: assembly of H2B K34ub modified and unmodified nucleosomes with increasing concentration of recombinant histone H1 0 .

    Techniques Used: SDS Page, Clear Native PAGE, Modification, Serial Dilution, Recombinant, Concentration Assay

    Eviction of histone dimer in H2B K34ub nucleosome by histone dimer acceptors. ( A ) Left panel: SDS-PAGE of NAP1. Middle, right panels: unmodified and H2B K34ub nucleosomes assembled on a 147 or 177 bp 601 DNA were post-assembly incubated with NAP1 for 2.5 h at 26 or 37°C in a buffer containing 100 mM NaCl. ( B ) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA for 2.5 h at 26/37°C in a buffer containing at 100 mM NaCl. ( C ) H2B K34ub nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA at 26/37°C for 2.5 h or at 26°C for 20 h (the ‘20 h’ and ‘2.5 h’ samples were resolved in different gels).
    Figure Legend Snippet: Eviction of histone dimer in H2B K34ub nucleosome by histone dimer acceptors. ( A ) Left panel: SDS-PAGE of NAP1. Middle, right panels: unmodified and H2B K34ub nucleosomes assembled on a 147 or 177 bp 601 DNA were post-assembly incubated with NAP1 for 2.5 h at 26 or 37°C in a buffer containing 100 mM NaCl. ( B ) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA for 2.5 h at 26/37°C in a buffer containing at 100 mM NaCl. ( C ) H2B K34ub nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA at 26/37°C for 2.5 h or at 26°C for 20 h (the ‘20 h’ and ‘2.5 h’ samples were resolved in different gels).

    Techniques Used: SDS Page, Incubation

    Stability of H2B K34ub- versus H2B K120ub-nucleosomes. ( A ) Left panel: SDS-PAGE of H2B K120ub and K34ub histone octamers. Right panel: unmodified and H2B K120ub/ K34ub nucleosomes assembled on 147 bp 601 DNA. ( B ) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA for 2.5 h at indicated temperature/ionic conditions. ( C ) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with NAP1 for 2.5 h at 26/37°C in a buffer containing at 140 mM NaCl.
    Figure Legend Snippet: Stability of H2B K34ub- versus H2B K120ub-nucleosomes. ( A ) Left panel: SDS-PAGE of H2B K120ub and K34ub histone octamers. Right panel: unmodified and H2B K120ub/ K34ub nucleosomes assembled on 147 bp 601 DNA. ( B ) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA for 2.5 h at indicated temperature/ionic conditions. ( C ) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with NAP1 for 2.5 h at 26/37°C in a buffer containing at 140 mM NaCl.

    Techniques Used: SDS Page, Incubation

    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

    Stability of H2B K34ub nucleosomes. ( A ) Native PAGE for nucleosome assembly with increasing amounts of unmodified and H2B K34ub histone octamers and their mixture at ratios indicated on top. Quantification was performed by Image J and relative abundance of each species was summarized in Table 1 . The representative result from three repeats was presented. ( B ) H2B K34ub (top panel) or Unmodified (bottom panel) nucleosomes were assembled on nicked, positively or negatively supercoiled plasmids containing 12 copies of 177 bp 601 DNA. After assembly 601 nucleosomes were released with ScaI and resolved on native PAGE. ( C ) The nucleosome assembly containing histones of indicated ratios (top) were incubated with competitor DNA for 2 h at 26°C in a buffer containing 200 mm NaCl. ( D ) Nucleosomes assembled on 147 bp 601 DNA (as indicated on top) were incubated with or without CM-50 Sephadex.
    Figure Legend Snippet: Stability of H2B K34ub nucleosomes. ( A ) Native PAGE for nucleosome assembly with increasing amounts of unmodified and H2B K34ub histone octamers and their mixture at ratios indicated on top. Quantification was performed by Image J and relative abundance of each species was summarized in Table 1 . The representative result from three repeats was presented. ( B ) H2B K34ub (top panel) or Unmodified (bottom panel) nucleosomes were assembled on nicked, positively or negatively supercoiled plasmids containing 12 copies of 177 bp 601 DNA. After assembly 601 nucleosomes were released with ScaI and resolved on native PAGE. ( C ) The nucleosome assembly containing histones of indicated ratios (top) were incubated with competitor DNA for 2 h at 26°C in a buffer containing 200 mm NaCl. ( D ) Nucleosomes assembled on 147 bp 601 DNA (as indicated on top) were incubated with or without CM-50 Sephadex.

    Techniques Used: Clear Native PAGE, Incubation

    Effects of underlying DNA sequence on stability of H2B K120ub and K34ub nucleosomes. ( A ) Nucleosomes assembled on 146 bp 5S DNA. ( B ) Assembled nucleosomes were incubated with competitor DNA for 2.5 h at 26°C in a buffer containing at 200 mM NaCl. ( C ) Nucleosomes incubated with competitor DNA for 2.5 h at indicated temperature/ ionic conditions. ( D and E ) Nucleosomes assembled with unmodified or H2B K34ub histones, or their 1/0.57 mixture, were post-assembly incubated for 2.5 h at 26 or 37°C with (D) competitor DNA in a buffer containing at 100 mM NaCl or (E) NAP1 in a buffer containing at 150 mM NaCl. Densitometry tracing of indicated gel lanes is shown at the bottom of gel images.
    Figure Legend Snippet: Effects of underlying DNA sequence on stability of H2B K120ub and K34ub nucleosomes. ( A ) Nucleosomes assembled on 146 bp 5S DNA. ( B ) Assembled nucleosomes were incubated with competitor DNA for 2.5 h at 26°C in a buffer containing at 200 mM NaCl. ( C ) Nucleosomes incubated with competitor DNA for 2.5 h at indicated temperature/ ionic conditions. ( D and E ) Nucleosomes assembled with unmodified or H2B K34ub histones, or their 1/0.57 mixture, were post-assembly incubated for 2.5 h at 26 or 37°C with (D) competitor DNA in a buffer containing at 100 mM NaCl or (E) NAP1 in a buffer containing at 150 mM NaCl. Densitometry tracing of indicated gel lanes is shown at the bottom of gel images.

    Techniques Used: Sequencing, Incubation

    3) Product Images from "Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer"

    Article Title: Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00787-16

    In vitro analysis of RpnA endonuclease activity. (A) WT RpnA cleaves pUC19, RpnA-D63A does not cleave pUC19, and RpnA-D165A is more active on pUC19. The pUC19 DNA (29 nM, 50 μg/ml) is initially supercoiled but can be relaxed by nicks, linearized by double-strand cleavage, or cleaved further. The supercoiled (control), relaxed (Nb.BtsI), and linear (HindIII) forms are indicated. pUC19 was treated with RpnA-inactive RpnA-D63A or hyperactive RpnA-D165A (15 μM, 45 min). (B) Time course of an RpnA (7.5 μM)-pUC19 (29 nM) digest. Band intensity was compared to determine the relative amounts of supercoiled, nicked, and linear pUC19 at each time point. Over 90% of the supercoiled pUC19 was digested within 180 min. (C) RpnA endonuclease activity depends on divalent cation and is stimulated by Ca 2+ . The reaction buffer was 50 mM NaCl and 10 mM Tris, pH 8.0; the indicated additives were at 10 mM each. RpnA at 3.8 μM was added for 18 h. (D) RpnA cleavage products provide a DNA polymerase primer. pUC19 was digested with RpnA, DNase I, or micrococcal nuclease (MNase) to produce similar smears and then incubated with fluorescein-labeled dNTPs and the Klenow fragment of DNA polymerase. DNA was visualized by ethidium bromide (EtBr; left) or fluorescein (middle), with the two signals being merged at the right. RpnA- and DNase I-digested DNAs were effectively labeled, but micrococcal nuclease-digested DNA was not.
    Figure Legend Snippet: In vitro analysis of RpnA endonuclease activity. (A) WT RpnA cleaves pUC19, RpnA-D63A does not cleave pUC19, and RpnA-D165A is more active on pUC19. The pUC19 DNA (29 nM, 50 μg/ml) is initially supercoiled but can be relaxed by nicks, linearized by double-strand cleavage, or cleaved further. The supercoiled (control), relaxed (Nb.BtsI), and linear (HindIII) forms are indicated. pUC19 was treated with RpnA-inactive RpnA-D63A or hyperactive RpnA-D165A (15 μM, 45 min). (B) Time course of an RpnA (7.5 μM)-pUC19 (29 nM) digest. Band intensity was compared to determine the relative amounts of supercoiled, nicked, and linear pUC19 at each time point. Over 90% of the supercoiled pUC19 was digested within 180 min. (C) RpnA endonuclease activity depends on divalent cation and is stimulated by Ca 2+ . The reaction buffer was 50 mM NaCl and 10 mM Tris, pH 8.0; the indicated additives were at 10 mM each. RpnA at 3.8 μM was added for 18 h. (D) RpnA cleavage products provide a DNA polymerase primer. pUC19 was digested with RpnA, DNase I, or micrococcal nuclease (MNase) to produce similar smears and then incubated with fluorescein-labeled dNTPs and the Klenow fragment of DNA polymerase. DNA was visualized by ethidium bromide (EtBr; left) or fluorescein (middle), with the two signals being merged at the right. RpnA- and DNase I-digested DNAs were effectively labeled, but micrococcal nuclease-digested DNA was not.

    Techniques Used: In Vitro, Activity Assay, Incubation, Labeling

    4) Product Images from "A plug and play microfluidic platform for standardized sensitive low-input Chromatin Immunoprecipitation"

    Article Title: A plug and play microfluidic platform for standardized sensitive low-input Chromatin Immunoprecipitation

    Journal: bioRxiv

    doi: 10.1101/2020.01.02.893180

    PnP-ChIP-seq using small cell quantities by the use of MNase shearing on 15,000 mESCs. (A) Gene-centered genome browser view for PnP-ChIP-Seq of H3K4me3, H3K4me1, H3K27ac and H3K36me3. (B) Overlap between de novo peak calls of PnP-ChIP-seq and bulk ChIP-Seq. (C) Heatmap of merged peak set for various starting amount of sonicated chromatin for PnP-ChIP-Seq of H3K4me3, H3K4me1, H3K27ac and H3K36me3. (D) Cross-correlations of PnP-ChIP-Seq using tag counts of merged peak set.
    Figure Legend Snippet: PnP-ChIP-seq using small cell quantities by the use of MNase shearing on 15,000 mESCs. (A) Gene-centered genome browser view for PnP-ChIP-Seq of H3K4me3, H3K4me1, H3K27ac and H3K36me3. (B) Overlap between de novo peak calls of PnP-ChIP-seq and bulk ChIP-Seq. (C) Heatmap of merged peak set for various starting amount of sonicated chromatin for PnP-ChIP-Seq of H3K4me3, H3K4me1, H3K27ac and H3K36me3. (D) Cross-correlations of PnP-ChIP-Seq using tag counts of merged peak set.

    Techniques Used: Chromatin Immunoprecipitation, Sonication

    5) Product Images from "EPIGENETIC ANALYSIS OF SV40 MINICHROMOSOMES"

    Article Title: EPIGENETIC ANALYSIS OF SV40 MINICHROMOSOMES

    Journal: Current protocols in microbiology

    doi: 10.1002/cpmc.35

    A comparison by agarose gel electrophoresis of crude SV40 virus before and after DNAse I digestion Crude SV40 virus was concentrated by centrifugation, resuspended in buffer and digested with DNAse I. For comparison purposes purified SV40 DNA prepared by the Hirt procedure was also analyzed. Lane 1, Hirt SV40 DNA, Lane 2, Crude concentrated SV40 before digestion, and Lane 3, Concentrated SV40 after DNAse I digestion. The positions of form I (supercoiled SV40 DNA0 and form II (relaxed SV40 DNA) are indicated on the side of the figure.
    Figure Legend Snippet: A comparison by agarose gel electrophoresis of crude SV40 virus before and after DNAse I digestion Crude SV40 virus was concentrated by centrifugation, resuspended in buffer and digested with DNAse I. For comparison purposes purified SV40 DNA prepared by the Hirt procedure was also analyzed. Lane 1, Hirt SV40 DNA, Lane 2, Crude concentrated SV40 before digestion, and Lane 3, Concentrated SV40 after DNAse I digestion. The positions of form I (supercoiled SV40 DNA0 and form II (relaxed SV40 DNA) are indicated on the side of the figure.

    Techniques Used: Agarose Gel Electrophoresis, Centrifugation, Purification

    Flowchart of Preparation of SV40 Chromatin and Subsequent Epigenetic Analysis
    Figure Legend Snippet: Flowchart of Preparation of SV40 Chromatin and Subsequent Epigenetic Analysis

    Techniques Used:

    Next Generation Sequencing analyses of micrococcal nuclease digested SV40 chromatin obtained from minichromosomes and virions Libraries were prepared from micrococcal nuclease digested SV40 minichromosomes obtained 48 hours post-infection (top) and from disrupted SV40 virions (bottom) using the protocols described in the text. The libraries were sequenced by paired-end sequencing using an Illumina MiSeq. Sequencing reads were plotted against the SV40 genome linearized between nucleotide number 5243 and 1. The sequencing reads from at least four biological replicates were then normalized and merged to generate the heatmap comparison shown. The intensity of the yellow color indicates the frequency in which a nucleosome was found located at a particular site in the SV40 genome with the brightest yellow corresponding to the highest frequency.
    Figure Legend Snippet: Next Generation Sequencing analyses of micrococcal nuclease digested SV40 chromatin obtained from minichromosomes and virions Libraries were prepared from micrococcal nuclease digested SV40 minichromosomes obtained 48 hours post-infection (top) and from disrupted SV40 virions (bottom) using the protocols described in the text. The libraries were sequenced by paired-end sequencing using an Illumina MiSeq. Sequencing reads were plotted against the SV40 genome linearized between nucleotide number 5243 and 1. The sequencing reads from at least four biological replicates were then normalized and merged to generate the heatmap comparison shown. The intensity of the yellow color indicates the frequency in which a nucleosome was found located at a particular site in the SV40 genome with the brightest yellow corresponding to the highest frequency.

    Techniques Used: Next-Generation Sequencing, Infection, Sequencing

    6) Product Images from "RNA-dependent chromatin targeting of TET2 for endogenous retrovirus control in pluripotent stem cells"

    Article Title: RNA-dependent chromatin targeting of TET2 for endogenous retrovirus control in pluripotent stem cells

    Journal: Nature genetics

    doi: 10.1038/s41588-018-0060-9

    PSPC1 and TET2 silence MERVL transcriptionally and post-transcriptionally a , MERVL expression in Tet1/2/3 triple knock-out ( Tet TKO) ESCs rescued with an empty vector (+EV), a wild-type (+TET2WT), or a catalytic mutant (+TET2Mut) TET2. Center line, median; box and whisker plots: ± 10th–90th percentile range. Data are from 5 independent experiments (n=14 total technical replicates for each rescue). Two-tailed Student’s t -test was applied. ns, not significant. b–c , MERVL and IAP enrichment, compared to U6 negative control, among anti-5hmC immunoprecipitated RNAs in Tet TKO (b) and Pspc1 KO (c) ESCs rescued with an empty vector (+EV), a wild-type, or a mutant TET2/PSPC1. Data are presented as mean ± s.e.m. (n=3 independent experiments). Two-tailed Student’s t -test was applied. ns, not significant. d , (Top) Schematic of the protocol used for inhibition of transcription with α-Amanitin for RNA stability assay. (Bottom) Relative abundance of MERVL RNA in Pspc1 WT an d KO ESCs after transcriptional inhibition for 1, 2, or 4 hours with α-Amanitin. Data are normalized to untreated cells at time 0 h (Vehicle without treatment). Error bars indicate s.e.m. (n=3). Two-tailed Student’s t -test was applied. ns, not significant. e , A model of MERVL regulation by PSPC1/TET2 and HDAC1/2 in ESCs. PSPC1 binding to actively transcribed MERVL RNAs recruits TET2 and HDAC1/2 to chromatin. TET2 catalyzes 5hmC modification of MERVL RNAs resulting in their destabilization, and HDAC1/2 deacetylate histones at the chromatin level leading to transcriptional repression of the MERVL loci. Transcriptional and posttranscriptional repression of MERVL leads to the release of the PSPC1-TET2-HDAC1/2 complex from chromatin. Sporadic reactivation of MERVL expression, well-recognized in conventionally cultured ESCs 10 , via a yet-to-be defined mechanism, leads to the recruitment PSPC1-TET2-HDAC1/2 for transcriptional and posttranscriptional control of MERVL and coordinated gene expression. Illustration by Jill Gregory. Printed with permission of ©Mount Sinai Health System.
    Figure Legend Snippet: PSPC1 and TET2 silence MERVL transcriptionally and post-transcriptionally a , MERVL expression in Tet1/2/3 triple knock-out ( Tet TKO) ESCs rescued with an empty vector (+EV), a wild-type (+TET2WT), or a catalytic mutant (+TET2Mut) TET2. Center line, median; box and whisker plots: ± 10th–90th percentile range. Data are from 5 independent experiments (n=14 total technical replicates for each rescue). Two-tailed Student’s t -test was applied. ns, not significant. b–c , MERVL and IAP enrichment, compared to U6 negative control, among anti-5hmC immunoprecipitated RNAs in Tet TKO (b) and Pspc1 KO (c) ESCs rescued with an empty vector (+EV), a wild-type, or a mutant TET2/PSPC1. Data are presented as mean ± s.e.m. (n=3 independent experiments). Two-tailed Student’s t -test was applied. ns, not significant. d , (Top) Schematic of the protocol used for inhibition of transcription with α-Amanitin for RNA stability assay. (Bottom) Relative abundance of MERVL RNA in Pspc1 WT an d KO ESCs after transcriptional inhibition for 1, 2, or 4 hours with α-Amanitin. Data are normalized to untreated cells at time 0 h (Vehicle without treatment). Error bars indicate s.e.m. (n=3). Two-tailed Student’s t -test was applied. ns, not significant. e , A model of MERVL regulation by PSPC1/TET2 and HDAC1/2 in ESCs. PSPC1 binding to actively transcribed MERVL RNAs recruits TET2 and HDAC1/2 to chromatin. TET2 catalyzes 5hmC modification of MERVL RNAs resulting in their destabilization, and HDAC1/2 deacetylate histones at the chromatin level leading to transcriptional repression of the MERVL loci. Transcriptional and posttranscriptional repression of MERVL leads to the release of the PSPC1-TET2-HDAC1/2 complex from chromatin. Sporadic reactivation of MERVL expression, well-recognized in conventionally cultured ESCs 10 , via a yet-to-be defined mechanism, leads to the recruitment PSPC1-TET2-HDAC1/2 for transcriptional and posttranscriptional control of MERVL and coordinated gene expression. Illustration by Jill Gregory. Printed with permission of ©Mount Sinai Health System.

    Techniques Used: Expressing, Knock-Out, Plasmid Preparation, Mutagenesis, Whisker Assay, Two Tailed Test, Negative Control, Immunoprecipitation, Inhibition, Stability Assay, Binding Assay, Modification, Cell Culture

    7) Product Images from "Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer"

    Article Title: Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00787-16

    In vitro analysis of RpnA endonuclease activity. (A) WT RpnA cleaves pUC19, RpnA-D63A does not cleave pUC19, and RpnA-D165A is more active on pUC19. The pUC19 DNA (29 nM, 50 μg/ml) is initially supercoiled but can be relaxed by nicks, linearized by double-strand cleavage, or cleaved further. The supercoiled (control), relaxed (Nb.BtsI), and linear (HindIII) forms are indicated. pUC19 was treated with RpnA-inactive RpnA-D63A or hyperactive RpnA-D165A (15 μM, 45 min). (B) Time course of an RpnA (7.5 μM)-pUC19 (29 nM) digest. Band intensity was compared to determine the relative amounts of supercoiled, nicked, and linear pUC19 at each time point. Over 90% of the supercoiled pUC19 was digested within 180 min. (C) RpnA endonuclease activity depends on divalent cation and is stimulated by Ca 2+ . The reaction buffer was 50 mM NaCl and 10 mM Tris, pH 8.0; the indicated additives were at 10 mM each. RpnA at 3.8 μM was added for 18 h. (D) RpnA cleavage products provide a DNA polymerase primer. pUC19 was digested with RpnA, DNase I, or micrococcal nuclease (MNase) to produce similar smears and then incubated with fluorescein-labeled dNTPs and the Klenow fragment of DNA polymerase. DNA was visualized by ethidium bromide (EtBr; left) or fluorescein (middle), with the two signals being merged at the right. RpnA- and DNase I-digested DNAs were effectively labeled, but micrococcal nuclease-digested DNA was not.
    Figure Legend Snippet: In vitro analysis of RpnA endonuclease activity. (A) WT RpnA cleaves pUC19, RpnA-D63A does not cleave pUC19, and RpnA-D165A is more active on pUC19. The pUC19 DNA (29 nM, 50 μg/ml) is initially supercoiled but can be relaxed by nicks, linearized by double-strand cleavage, or cleaved further. The supercoiled (control), relaxed (Nb.BtsI), and linear (HindIII) forms are indicated. pUC19 was treated with RpnA-inactive RpnA-D63A or hyperactive RpnA-D165A (15 μM, 45 min). (B) Time course of an RpnA (7.5 μM)-pUC19 (29 nM) digest. Band intensity was compared to determine the relative amounts of supercoiled, nicked, and linear pUC19 at each time point. Over 90% of the supercoiled pUC19 was digested within 180 min. (C) RpnA endonuclease activity depends on divalent cation and is stimulated by Ca 2+ . The reaction buffer was 50 mM NaCl and 10 mM Tris, pH 8.0; the indicated additives were at 10 mM each. RpnA at 3.8 μM was added for 18 h. (D) RpnA cleavage products provide a DNA polymerase primer. pUC19 was digested with RpnA, DNase I, or micrococcal nuclease (MNase) to produce similar smears and then incubated with fluorescein-labeled dNTPs and the Klenow fragment of DNA polymerase. DNA was visualized by ethidium bromide (EtBr; left) or fluorescein (middle), with the two signals being merged at the right. RpnA- and DNase I-digested DNAs were effectively labeled, but micrococcal nuclease-digested DNA was not.

    Techniques Used: In Vitro, Activity Assay, Incubation, Labeling

    8) Product Images from "EPIGENETIC ANALYSIS OF SV40 MINICHROMOSOMES"

    Article Title: EPIGENETIC ANALYSIS OF SV40 MINICHROMOSOMES

    Journal: Current protocols in microbiology

    doi: 10.1002/cpmc.35

    A comparison by agarose gel electrophoresis of crude SV40 virus before and after DNAse I digestion Crude SV40 virus was concentrated by centrifugation, resuspended in buffer and digested with DNAse I. For comparison purposes purified SV40 DNA prepared by the Hirt procedure was also analyzed. Lane 1, Hirt SV40 DNA, Lane 2, Crude concentrated SV40 before digestion, and Lane 3, Concentrated SV40 after DNAse I digestion. The positions of form I (supercoiled SV40 DNA0 and form II (relaxed SV40 DNA) are indicated on the side of the figure.
    Figure Legend Snippet: A comparison by agarose gel electrophoresis of crude SV40 virus before and after DNAse I digestion Crude SV40 virus was concentrated by centrifugation, resuspended in buffer and digested with DNAse I. For comparison purposes purified SV40 DNA prepared by the Hirt procedure was also analyzed. Lane 1, Hirt SV40 DNA, Lane 2, Crude concentrated SV40 before digestion, and Lane 3, Concentrated SV40 after DNAse I digestion. The positions of form I (supercoiled SV40 DNA0 and form II (relaxed SV40 DNA) are indicated on the side of the figure.

    Techniques Used: Agarose Gel Electrophoresis, Centrifugation, Purification

    Flowchart of Preparation of SV40 Chromatin and Subsequent Epigenetic Analysis
    Figure Legend Snippet: Flowchart of Preparation of SV40 Chromatin and Subsequent Epigenetic Analysis

    Techniques Used:

    Next Generation Sequencing analyses of micrococcal nuclease digested SV40 chromatin obtained from minichromosomes and virions Libraries were prepared from micrococcal nuclease digested SV40 minichromosomes obtained 48 hours post-infection (top) and from disrupted SV40 virions (bottom) using the protocols described in the text. The libraries were sequenced by paired-end sequencing using an Illumina MiSeq. Sequencing reads were plotted against the SV40 genome linearized between nucleotide number 5243 and 1. The sequencing reads from at least four biological replicates were then normalized and merged to generate the heatmap comparison shown. The intensity of the yellow color indicates the frequency in which a nucleosome was found located at a particular site in the SV40 genome with the brightest yellow corresponding to the highest frequency.
    Figure Legend Snippet: Next Generation Sequencing analyses of micrococcal nuclease digested SV40 chromatin obtained from minichromosomes and virions Libraries were prepared from micrococcal nuclease digested SV40 minichromosomes obtained 48 hours post-infection (top) and from disrupted SV40 virions (bottom) using the protocols described in the text. The libraries were sequenced by paired-end sequencing using an Illumina MiSeq. Sequencing reads were plotted against the SV40 genome linearized between nucleotide number 5243 and 1. The sequencing reads from at least four biological replicates were then normalized and merged to generate the heatmap comparison shown. The intensity of the yellow color indicates the frequency in which a nucleosome was found located at a particular site in the SV40 genome with the brightest yellow corresponding to the highest frequency.

    Techniques Used: Next-Generation Sequencing, Infection, Sequencing

    9) Product Images from "RNA-dependent chromatin targeting of TET2 for endogenous retrovirus control in pluripotent stem cells"

    Article Title: RNA-dependent chromatin targeting of TET2 for endogenous retrovirus control in pluripotent stem cells

    Journal: Nature genetics

    doi: 10.1038/s41588-018-0060-9

    PSPC1 and TET2 silence MERVL transcriptionally and post-transcriptionally a , MERVL expression in Tet1/2/3 triple knock-out ( Tet TKO) ESCs rescued with an empty vector (+EV), a wild-type (+TET2WT), or a catalytic mutant (+TET2Mut) TET2. Center line, median; box and whisker plots: ± 10th–90th percentile range. Data are from 5 independent experiments (n=14 total technical replicates for each rescue). Two-tailed Student’s t -test was applied. ns, not significant. b–c , MERVL and IAP enrichment, compared to U6 negative control, among anti-5hmC immunoprecipitated RNAs in Tet TKO (b) and Pspc1 KO (c) ESCs rescued with an empty vector (+EV), a wild-type, or a mutant TET2/PSPC1. Data are presented as mean ± s.e.m. (n=3 independent experiments). Two-tailed Student’s t -test was applied. ns, not significant. d , (Top) Schematic of the protocol used for inhibition of transcription with α-Amanitin for RNA stability assay. (Bottom) Relative abundance of MERVL RNA in Pspc1 WT an d KO ESCs after transcriptional inhibition for 1, 2, or 4 hours with α-Amanitin. Data are normalized to untreated cells at time 0 h (Vehicle without treatment). Error bars indicate s.e.m. (n=3). Two-tailed Student’s t -test was applied. ns, not significant. e , A model of MERVL regulation by PSPC1/TET2 and HDAC1/2 in ESCs. PSPC1 binding to actively transcribed MERVL RNAs recruits TET2 and HDAC1/2 to chromatin. TET2 catalyzes 5hmC modification of MERVL RNAs resulting in their destabilization, and HDAC1/2 deacetylate histones at the chromatin level leading to transcriptional repression of the MERVL loci. Transcriptional and posttranscriptional repression of MERVL leads to the release of the PSPC1-TET2-HDAC1/2 complex from chromatin. Sporadic reactivation of MERVL , via a yet-to-be defined mechanism, leads to the recruitment PSPC1-TET2-HDAC1/2 for transcriptional and posttranscriptional control of MERVL and coordinated gene expression. Illustration by Jill Gregory. Printed with permission of ©Mount Sinai Health System.
    Figure Legend Snippet: PSPC1 and TET2 silence MERVL transcriptionally and post-transcriptionally a , MERVL expression in Tet1/2/3 triple knock-out ( Tet TKO) ESCs rescued with an empty vector (+EV), a wild-type (+TET2WT), or a catalytic mutant (+TET2Mut) TET2. Center line, median; box and whisker plots: ± 10th–90th percentile range. Data are from 5 independent experiments (n=14 total technical replicates for each rescue). Two-tailed Student’s t -test was applied. ns, not significant. b–c , MERVL and IAP enrichment, compared to U6 negative control, among anti-5hmC immunoprecipitated RNAs in Tet TKO (b) and Pspc1 KO (c) ESCs rescued with an empty vector (+EV), a wild-type, or a mutant TET2/PSPC1. Data are presented as mean ± s.e.m. (n=3 independent experiments). Two-tailed Student’s t -test was applied. ns, not significant. d , (Top) Schematic of the protocol used for inhibition of transcription with α-Amanitin for RNA stability assay. (Bottom) Relative abundance of MERVL RNA in Pspc1 WT an d KO ESCs after transcriptional inhibition for 1, 2, or 4 hours with α-Amanitin. Data are normalized to untreated cells at time 0 h (Vehicle without treatment). Error bars indicate s.e.m. (n=3). Two-tailed Student’s t -test was applied. ns, not significant. e , A model of MERVL regulation by PSPC1/TET2 and HDAC1/2 in ESCs. PSPC1 binding to actively transcribed MERVL RNAs recruits TET2 and HDAC1/2 to chromatin. TET2 catalyzes 5hmC modification of MERVL RNAs resulting in their destabilization, and HDAC1/2 deacetylate histones at the chromatin level leading to transcriptional repression of the MERVL loci. Transcriptional and posttranscriptional repression of MERVL leads to the release of the PSPC1-TET2-HDAC1/2 complex from chromatin. Sporadic reactivation of MERVL , via a yet-to-be defined mechanism, leads to the recruitment PSPC1-TET2-HDAC1/2 for transcriptional and posttranscriptional control of MERVL and coordinated gene expression. Illustration by Jill Gregory. Printed with permission of ©Mount Sinai Health System.

    Techniques Used: Expressing, Knock-Out, Plasmid Preparation, Mutagenesis, Whisker Assay, Two Tailed Test, Negative Control, Immunoprecipitation, Inhibition, Stability Assay, Binding Assay, Modification

    10) Product Images from "Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer"

    Article Title: Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00787-16

    In vitro analysis of RpnA endonuclease activity. (A) WT RpnA cleaves pUC19, RpnA-D63A does not cleave pUC19, and RpnA-D165A is more active on pUC19. The pUC19 DNA (29 nM, 50 μg/ml) is initially supercoiled but can be relaxed by nicks, linearized by double-strand cleavage, or cleaved further. The supercoiled (control), relaxed (Nb.BtsI), and linear (HindIII) forms are indicated. pUC19 was treated with RpnA-inactive RpnA-D63A or hyperactive RpnA-D165A (15 μM, 45 min). (B) Time course of an RpnA (7.5 μM)-pUC19 (29 nM) digest. Band intensity was compared to determine the relative amounts of supercoiled, nicked, and linear pUC19 at each time point. Over 90% of the supercoiled pUC19 was digested within 180 min. (C) RpnA endonuclease activity depends on divalent cation and is stimulated by Ca 2+ . The reaction buffer was 50 mM NaCl and 10 mM Tris, pH 8.0; the indicated additives were at 10 mM each. RpnA at 3.8 μM was added for 18 h. (D) RpnA cleavage products provide a DNA polymerase primer. pUC19 was digested with RpnA, DNase I, or micrococcal nuclease (MNase) to produce similar smears and then incubated with fluorescein-labeled dNTPs and the Klenow fragment of DNA polymerase. DNA was visualized by ethidium bromide (EtBr; left) or fluorescein (middle), with the two signals being merged at the right. RpnA- and DNase I-digested DNAs were effectively labeled, but micrococcal nuclease-digested DNA was not.
    Figure Legend Snippet: In vitro analysis of RpnA endonuclease activity. (A) WT RpnA cleaves pUC19, RpnA-D63A does not cleave pUC19, and RpnA-D165A is more active on pUC19. The pUC19 DNA (29 nM, 50 μg/ml) is initially supercoiled but can be relaxed by nicks, linearized by double-strand cleavage, or cleaved further. The supercoiled (control), relaxed (Nb.BtsI), and linear (HindIII) forms are indicated. pUC19 was treated with RpnA-inactive RpnA-D63A or hyperactive RpnA-D165A (15 μM, 45 min). (B) Time course of an RpnA (7.5 μM)-pUC19 (29 nM) digest. Band intensity was compared to determine the relative amounts of supercoiled, nicked, and linear pUC19 at each time point. Over 90% of the supercoiled pUC19 was digested within 180 min. (C) RpnA endonuclease activity depends on divalent cation and is stimulated by Ca 2+ . The reaction buffer was 50 mM NaCl and 10 mM Tris, pH 8.0; the indicated additives were at 10 mM each. RpnA at 3.8 μM was added for 18 h. (D) RpnA cleavage products provide a DNA polymerase primer. pUC19 was digested with RpnA, DNase I, or micrococcal nuclease (MNase) to produce similar smears and then incubated with fluorescein-labeled dNTPs and the Klenow fragment of DNA polymerase. DNA was visualized by ethidium bromide (EtBr; left) or fluorescein (middle), with the two signals being merged at the right. RpnA- and DNase I-digested DNAs were effectively labeled, but micrococcal nuclease-digested DNA was not.

    Techniques Used: In Vitro, Activity Assay, Incubation, Labeling

    Distribution of genomic exchanges in recombinants. (A) Diagram of markers that distinguish the donor and recipient genomes and distances from the selected tetRA cassette. The chromosome segregation site dif is also shown. ICR, restriction enzyme gene cluster known as the immigration control region. (B) Recombinants were screened for the cat , npt , mrr , fhuA , and lacZ markers. Recombinants containing both tetRA and mrr were classified as having genome additions, and the results for these recombinants are not shown. Horizontal bars indicate the extent of donor DNA (orange) that we inferred replaced the recipient genome (blue) during the recombination event. NA, not available. (C) Proportion of recombinants in each class from a basal mating (WT; ER3435 × ER3473) with or without AZT treatment or from a mating in which rpnA was overexpressed (RpnA; ER3435 × ER3514). For the WT, most recombinants were created by large replacements of over 400 kb of genomic DNA. In the RpnA and AZT matings, large replacements were less frequent, and over half of the genomic replacements were within the 236-kb segment between the npt and fhuA markers flanking the selected marker, tetRA .
    Figure Legend Snippet: Distribution of genomic exchanges in recombinants. (A) Diagram of markers that distinguish the donor and recipient genomes and distances from the selected tetRA cassette. The chromosome segregation site dif is also shown. ICR, restriction enzyme gene cluster known as the immigration control region. (B) Recombinants were screened for the cat , npt , mrr , fhuA , and lacZ markers. Recombinants containing both tetRA and mrr were classified as having genome additions, and the results for these recombinants are not shown. Horizontal bars indicate the extent of donor DNA (orange) that we inferred replaced the recipient genome (blue) during the recombination event. NA, not available. (C) Proportion of recombinants in each class from a basal mating (WT; ER3435 × ER3473) with or without AZT treatment or from a mating in which rpnA was overexpressed (RpnA; ER3435 × ER3514). For the WT, most recombinants were created by large replacements of over 400 kb of genomic DNA. In the RpnA and AZT matings, large replacements were less frequent, and over half of the genomic replacements were within the 236-kb segment between the npt and fhuA markers flanking the selected marker, tetRA .

    Techniques Used: Marker

    11) Product Images from "ARGONAUTE2 cooperates with SWI/SNF complex to determine nucleosome occupancy at human Transcription Start Sites"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku1387

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

    Techniques Used: Transfection, Western Blot

    12) Product Images from "A plug and play microfluidic platform for standardized sensitive low-input Chromatin Immunoprecipitation"

    Article Title: A plug and play microfluidic platform for standardized sensitive low-input Chromatin Immunoprecipitation

    Journal: bioRxiv

    doi: 10.1101/2020.01.02.893180

    PnP-ChIP-seq using small quantites of bulk-sonicated crosslinked chromatin. (A) Genecentered genome browser view for PnP-ChIP-Seq of H3K4me3, H3K4me1, H3K27ac and H3K36me3. (B) Heatmap of merged peak set for various starting amount of sonicated chromatin for PnP-ChIP-Seq of H3K4me3, H3K4me1, H3K27ac and H3K36me3. (C) Cross-correlations of PnP-ChIP-Seq using tag counts of merged peak set. (D) Overlap between de novo peak calls of PnP-ChIP-seq and bulk ChIP-Seq. (E-F) Overlap between de novo peak calls of PnP-ChIP-seq.
    Figure Legend Snippet: PnP-ChIP-seq using small quantites of bulk-sonicated crosslinked chromatin. (A) Genecentered genome browser view for PnP-ChIP-Seq of H3K4me3, H3K4me1, H3K27ac and H3K36me3. (B) Heatmap of merged peak set for various starting amount of sonicated chromatin for PnP-ChIP-Seq of H3K4me3, H3K4me1, H3K27ac and H3K36me3. (C) Cross-correlations of PnP-ChIP-Seq using tag counts of merged peak set. (D) Overlap between de novo peak calls of PnP-ChIP-seq and bulk ChIP-Seq. (E-F) Overlap between de novo peak calls of PnP-ChIP-seq.

    Techniques Used: Chromatin Immunoprecipitation, Sonication

    Related Articles

    Isolation:

    Article Title: ARGONAUTE2 cooperates with SWI/SNF complex to determine nucleosome occupancy at human Transcription Start Sites
    Article Snippet: .. Isolation of nucleosomal DNA by micrococcal nuclease (MNase) digestion Digestion of chromatin from untreated, siCTRL- or siAGO2-treated HeLa S3 cells (2 × 106 ) was performed with 50 U of MNase (New England Biolabs) in 300 μl of permeabilization buffer (15 mM Tris–HCl pH 7.4, 300 mM sucrose, 60 mM KCl, 15 mM NaCl, 4 mM CaCl2 , 0.5 mM EGTA, 0.2% NP-40, 0.5 mM β-mercaptoethanol) for 20 min at 37°C. ..

    Incubation:

    Article Title: A peptide-based viral inactivator inhibits Zika virus infection in pregnant mice and fetuses
    Article Snippet: .. Briefly, about 1 × 103 plaque-forming units (p.f.u.) of ZIKV was incubated with Z2 or Z2-scr at room temperature for 2 h. The released genomic RNA from the treated ZIKV particles was then digested with micrococcal nuclease (New England BioLabs, MA) at 37 °C for 1 h. After inactivation of the residual RNase, the undigested genomic RNA in the intact viral particles was extracted using the Qiagen QIAamp Viral RNA Mini Kit (Valencia, CA) and reversed by using RT Reagent Kit (Takara Bio, Shiga, Japan). .. ZIKV RNA genome was quantified by SYBR PremixExTaqII (TliRNase H Plus from Takara Bio) and the Master Cycler Ep Realplex PCR System (Eppendorf, Hamburg, Germany) according to the manufacturers’ instructions.

    Sonication:

    Article Title: The orphan nuclear receptor NR4A2 is part of a p53–microRNA-34 network
    Article Snippet: .. Cell extracts were digested for 10 min with 50 units of micrococcal nuclease (New England Biolabs, Ipswich, MA) at 37 °C and further sonicated to yield sheared DNA fragments with an average length of 200 to 1000 base pairs. .. The sonicated samples were centrifuged to pellet the cell debris, and the supernatant was diluted 7-fold with ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl, and protease inhibitor cocktail).

    Sequencing:

    Article Title: EPIGENETIC ANALYSIS OF SV40 MINICHROMOSOMES
    Article Snippet: .. We presently prepare our libraries for sequencing from micrococcal nuclease digested SV40 chromatin and ChIP analyses using an NEBNEXT kit designed to work with the Illumina sequencing system. ..

    Lysis:

    Article Title: Analysis of neonatal brain lacking ATRX or MeCP2 reveals changes in nucleosome density, CTCF binding and chromatin looping
    Article Snippet: .. Cells were resuspended in lysis buffer [0.34 M sucrose, 60 mM KCl, 15 mM Tris–HCl, 15 mM NaCl, 0.5% NP-40 and 1× protease inhibitors (Sigma-Aldrich)] and flash-frozen and thawed three times, nuclei were centrifuged and resuspended in micrococcal nuclease digestion buffer (NEB). .. Micrococcal nuclease (2 U; NEB) was added and incubated at 37°C for 5 min, then quenched with EDTA.

    Chromatin Immunoprecipitation:

    Article Title: EPIGENETIC ANALYSIS OF SV40 MINICHROMOSOMES
    Article Snippet: .. We presently prepare our libraries for sequencing from micrococcal nuclease digested SV40 chromatin and ChIP analyses using an NEBNEXT kit designed to work with the Illumina sequencing system. ..

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs micrococcal nuclease digestion assembled nucleosome
    Assembly of H2B K34ub and unmodified <t>nucleosomes.</t> ( A ) Left panel: SDS-PAGE of H2B K34ub- (lane 1) and unmodified (lane 2) histone octamers. Right panel: native PAGE of modified nucleosomes assembled on 147 bp 601 DNA using increasing amounts histones. ( B ) SDS-PAGE of faster- (lane 1) and slower- (lane 2) migrating H2B K34ub assembly products extracted from the native gel in A. The densitometric tracing of the gel lanes is shown on the right. Quantification, by ImageJ, is normalized to that of the histone H4, which is arbitrarily set as 1. ( C ) Native PAGE for samples prepared by serial dilution of 2M NaCl mixtures of 147 bp 601 DNA and modified or unmodified histones to 1 M NaCl. ( D ) Left panel: SDS-PAGE of recombinant linker histone H1 0 . Right panel: assembly of H2B K34ub modified and unmodified nucleosomes with increasing concentration of recombinant histone H1 0 .
    Micrococcal Nuclease Digestion Assembled Nucleosome, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/micrococcal nuclease digestion assembled nucleosome/product/New England Biolabs
    Average 99 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    micrococcal nuclease digestion assembled nucleosome - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    Image Search Results


    Assembly of H2B K34ub and unmodified nucleosomes. ( A ) Left panel: SDS-PAGE of H2B K34ub- (lane 1) and unmodified (lane 2) histone octamers. Right panel: native PAGE of modified nucleosomes assembled on 147 bp 601 DNA using increasing amounts histones. ( B ) SDS-PAGE of faster- (lane 1) and slower- (lane 2) migrating H2B K34ub assembly products extracted from the native gel in A. The densitometric tracing of the gel lanes is shown on the right. Quantification, by ImageJ, is normalized to that of the histone H4, which is arbitrarily set as 1. ( C ) Native PAGE for samples prepared by serial dilution of 2M NaCl mixtures of 147 bp 601 DNA and modified or unmodified histones to 1 M NaCl. ( D ) Left panel: SDS-PAGE of recombinant linker histone H1 0 . Right panel: assembly of H2B K34ub modified and unmodified nucleosomes with increasing concentration of recombinant histone H1 0 .

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gky526

    Figure Lengend Snippet: Assembly of H2B K34ub and unmodified nucleosomes. ( A ) Left panel: SDS-PAGE of H2B K34ub- (lane 1) and unmodified (lane 2) histone octamers. Right panel: native PAGE of modified nucleosomes assembled on 147 bp 601 DNA using increasing amounts histones. ( B ) SDS-PAGE of faster- (lane 1) and slower- (lane 2) migrating H2B K34ub assembly products extracted from the native gel in A. The densitometric tracing of the gel lanes is shown on the right. Quantification, by ImageJ, is normalized to that of the histone H4, which is arbitrarily set as 1. ( C ) Native PAGE for samples prepared by serial dilution of 2M NaCl mixtures of 147 bp 601 DNA and modified or unmodified histones to 1 M NaCl. ( D ) Left panel: SDS-PAGE of recombinant linker histone H1 0 . Right panel: assembly of H2B K34ub modified and unmodified nucleosomes with increasing concentration of recombinant histone H1 0 .

    Article Snippet: Micrococcal nuclease digestion Assembled nucleosome (∼30 ng DNA) were digested with increasing amounts (typically 0.3/1/3/10/30/100 Kunitz units) of MNase (NEB) for 20 min at 26 or 37°C.

    Techniques: SDS Page, Clear Native PAGE, Modification, Serial Dilution, Recombinant, Concentration Assay

    Eviction of histone dimer in H2B K34ub nucleosome by histone dimer acceptors. ( A ) Left panel: SDS-PAGE of NAP1. Middle, right panels: unmodified and H2B K34ub nucleosomes assembled on a 147 or 177 bp 601 DNA were post-assembly incubated with NAP1 for 2.5 h at 26 or 37°C in a buffer containing 100 mM NaCl. ( B ) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA for 2.5 h at 26/37°C in a buffer containing at 100 mM NaCl. ( C ) H2B K34ub nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA at 26/37°C for 2.5 h or at 26°C for 20 h (the ‘20 h’ and ‘2.5 h’ samples were resolved in different gels).

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gky526

    Figure Lengend Snippet: Eviction of histone dimer in H2B K34ub nucleosome by histone dimer acceptors. ( A ) Left panel: SDS-PAGE of NAP1. Middle, right panels: unmodified and H2B K34ub nucleosomes assembled on a 147 or 177 bp 601 DNA were post-assembly incubated with NAP1 for 2.5 h at 26 or 37°C in a buffer containing 100 mM NaCl. ( B ) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA for 2.5 h at 26/37°C in a buffer containing at 100 mM NaCl. ( C ) H2B K34ub nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA at 26/37°C for 2.5 h or at 26°C for 20 h (the ‘20 h’ and ‘2.5 h’ samples were resolved in different gels).

    Article Snippet: Micrococcal nuclease digestion Assembled nucleosome (∼30 ng DNA) were digested with increasing amounts (typically 0.3/1/3/10/30/100 Kunitz units) of MNase (NEB) for 20 min at 26 or 37°C.

    Techniques: SDS Page, Incubation

    Stability of H2B K34ub- versus H2B K120ub-nucleosomes. ( A ) Left panel: SDS-PAGE of H2B K120ub and K34ub histone octamers. Right panel: unmodified and H2B K120ub/ K34ub nucleosomes assembled on 147 bp 601 DNA. ( B ) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA for 2.5 h at indicated temperature/ionic conditions. ( C ) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with NAP1 for 2.5 h at 26/37°C in a buffer containing at 140 mM NaCl.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gky526

    Figure Lengend Snippet: Stability of H2B K34ub- versus H2B K120ub-nucleosomes. ( A ) Left panel: SDS-PAGE of H2B K120ub and K34ub histone octamers. Right panel: unmodified and H2B K120ub/ K34ub nucleosomes assembled on 147 bp 601 DNA. ( B ) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA for 2.5 h at indicated temperature/ionic conditions. ( C ) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with NAP1 for 2.5 h at 26/37°C in a buffer containing at 140 mM NaCl.

    Article Snippet: Micrococcal nuclease digestion Assembled nucleosome (∼30 ng DNA) were digested with increasing amounts (typically 0.3/1/3/10/30/100 Kunitz units) of MNase (NEB) for 20 min at 26 or 37°C.

    Techniques: SDS Page, Incubation

    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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gky526

    Figure Lengend 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.

    Article Snippet: Micrococcal nuclease digestion Assembled nucleosome (∼30 ng DNA) were digested with increasing amounts (typically 0.3/1/3/10/30/100 Kunitz units) of MNase (NEB) for 20 min at 26 or 37°C.

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

    Stability of H2B K34ub nucleosomes. ( A ) Native PAGE for nucleosome assembly with increasing amounts of unmodified and H2B K34ub histone octamers and their mixture at ratios indicated on top. Quantification was performed by Image J and relative abundance of each species was summarized in Table 1 . The representative result from three repeats was presented. ( B ) H2B K34ub (top panel) or Unmodified (bottom panel) nucleosomes were assembled on nicked, positively or negatively supercoiled plasmids containing 12 copies of 177 bp 601 DNA. After assembly 601 nucleosomes were released with ScaI and resolved on native PAGE. ( C ) The nucleosome assembly containing histones of indicated ratios (top) were incubated with competitor DNA for 2 h at 26°C in a buffer containing 200 mm NaCl. ( D ) Nucleosomes assembled on 147 bp 601 DNA (as indicated on top) were incubated with or without CM-50 Sephadex.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gky526

    Figure Lengend Snippet: Stability of H2B K34ub nucleosomes. ( A ) Native PAGE for nucleosome assembly with increasing amounts of unmodified and H2B K34ub histone octamers and their mixture at ratios indicated on top. Quantification was performed by Image J and relative abundance of each species was summarized in Table 1 . The representative result from three repeats was presented. ( B ) H2B K34ub (top panel) or Unmodified (bottom panel) nucleosomes were assembled on nicked, positively or negatively supercoiled plasmids containing 12 copies of 177 bp 601 DNA. After assembly 601 nucleosomes were released with ScaI and resolved on native PAGE. ( C ) The nucleosome assembly containing histones of indicated ratios (top) were incubated with competitor DNA for 2 h at 26°C in a buffer containing 200 mm NaCl. ( D ) Nucleosomes assembled on 147 bp 601 DNA (as indicated on top) were incubated with or without CM-50 Sephadex.

    Article Snippet: Micrococcal nuclease digestion Assembled nucleosome (∼30 ng DNA) were digested with increasing amounts (typically 0.3/1/3/10/30/100 Kunitz units) of MNase (NEB) for 20 min at 26 or 37°C.

    Techniques: Clear Native PAGE, Incubation

    Effects of underlying DNA sequence on stability of H2B K120ub and K34ub nucleosomes. ( A ) Nucleosomes assembled on 146 bp 5S DNA. ( B ) Assembled nucleosomes were incubated with competitor DNA for 2.5 h at 26°C in a buffer containing at 200 mM NaCl. ( C ) Nucleosomes incubated with competitor DNA for 2.5 h at indicated temperature/ ionic conditions. ( D and E ) Nucleosomes assembled with unmodified or H2B K34ub histones, or their 1/0.57 mixture, were post-assembly incubated for 2.5 h at 26 or 37°C with (D) competitor DNA in a buffer containing at 100 mM NaCl or (E) NAP1 in a buffer containing at 150 mM NaCl. Densitometry tracing of indicated gel lanes is shown at the bottom of gel images.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gky526

    Figure Lengend Snippet: Effects of underlying DNA sequence on stability of H2B K120ub and K34ub nucleosomes. ( A ) Nucleosomes assembled on 146 bp 5S DNA. ( B ) Assembled nucleosomes were incubated with competitor DNA for 2.5 h at 26°C in a buffer containing at 200 mM NaCl. ( C ) Nucleosomes incubated with competitor DNA for 2.5 h at indicated temperature/ ionic conditions. ( D and E ) Nucleosomes assembled with unmodified or H2B K34ub histones, or their 1/0.57 mixture, were post-assembly incubated for 2.5 h at 26 or 37°C with (D) competitor DNA in a buffer containing at 100 mM NaCl or (E) NAP1 in a buffer containing at 150 mM NaCl. Densitometry tracing of indicated gel lanes is shown at the bottom of gel images.

    Article Snippet: Micrococcal nuclease digestion Assembled nucleosome (∼30 ng DNA) were digested with increasing amounts (typically 0.3/1/3/10/30/100 Kunitz units) of MNase (NEB) for 20 min at 26 or 37°C.

    Techniques: Sequencing, Incubation

    A comparison by agarose gel electrophoresis of crude SV40 virus before and after DNAse I digestion Crude SV40 virus was concentrated by centrifugation, resuspended in buffer and digested with DNAse I. For comparison purposes purified SV40 DNA prepared by the Hirt procedure was also analyzed. Lane 1, Hirt SV40 DNA, Lane 2, Crude concentrated SV40 before digestion, and Lane 3, Concentrated SV40 after DNAse I digestion. The positions of form I (supercoiled SV40 DNA0 and form II (relaxed SV40 DNA) are indicated on the side of the figure.

    Journal: Current protocols in microbiology

    Article Title: EPIGENETIC ANALYSIS OF SV40 MINICHROMOSOMES

    doi: 10.1002/cpmc.35

    Figure Lengend Snippet: A comparison by agarose gel electrophoresis of crude SV40 virus before and after DNAse I digestion Crude SV40 virus was concentrated by centrifugation, resuspended in buffer and digested with DNAse I. For comparison purposes purified SV40 DNA prepared by the Hirt procedure was also analyzed. Lane 1, Hirt SV40 DNA, Lane 2, Crude concentrated SV40 before digestion, and Lane 3, Concentrated SV40 after DNAse I digestion. The positions of form I (supercoiled SV40 DNA0 and form II (relaxed SV40 DNA) are indicated on the side of the figure.

    Article Snippet: We presently prepare our libraries for sequencing from micrococcal nuclease digested SV40 chromatin and ChIP analyses using an NEBNEXT kit designed to work with the Illumina sequencing system.

    Techniques: Agarose Gel Electrophoresis, Centrifugation, Purification

    Flowchart of Preparation of SV40 Chromatin and Subsequent Epigenetic Analysis

    Journal: Current protocols in microbiology

    Article Title: EPIGENETIC ANALYSIS OF SV40 MINICHROMOSOMES

    doi: 10.1002/cpmc.35

    Figure Lengend Snippet: Flowchart of Preparation of SV40 Chromatin and Subsequent Epigenetic Analysis

    Article Snippet: We presently prepare our libraries for sequencing from micrococcal nuclease digested SV40 chromatin and ChIP analyses using an NEBNEXT kit designed to work with the Illumina sequencing system.

    Techniques:

    Next Generation Sequencing analyses of micrococcal nuclease digested SV40 chromatin obtained from minichromosomes and virions Libraries were prepared from micrococcal nuclease digested SV40 minichromosomes obtained 48 hours post-infection (top) and from disrupted SV40 virions (bottom) using the protocols described in the text. The libraries were sequenced by paired-end sequencing using an Illumina MiSeq. Sequencing reads were plotted against the SV40 genome linearized between nucleotide number 5243 and 1. The sequencing reads from at least four biological replicates were then normalized and merged to generate the heatmap comparison shown. The intensity of the yellow color indicates the frequency in which a nucleosome was found located at a particular site in the SV40 genome with the brightest yellow corresponding to the highest frequency.

    Journal: Current protocols in microbiology

    Article Title: EPIGENETIC ANALYSIS OF SV40 MINICHROMOSOMES

    doi: 10.1002/cpmc.35

    Figure Lengend Snippet: Next Generation Sequencing analyses of micrococcal nuclease digested SV40 chromatin obtained from minichromosomes and virions Libraries were prepared from micrococcal nuclease digested SV40 minichromosomes obtained 48 hours post-infection (top) and from disrupted SV40 virions (bottom) using the protocols described in the text. The libraries were sequenced by paired-end sequencing using an Illumina MiSeq. Sequencing reads were plotted against the SV40 genome linearized between nucleotide number 5243 and 1. The sequencing reads from at least four biological replicates were then normalized and merged to generate the heatmap comparison shown. The intensity of the yellow color indicates the frequency in which a nucleosome was found located at a particular site in the SV40 genome with the brightest yellow corresponding to the highest frequency.

    Article Snippet: We presently prepare our libraries for sequencing from micrococcal nuclease digested SV40 chromatin and ChIP analyses using an NEBNEXT kit designed to work with the Illumina sequencing system.

    Techniques: Next-Generation Sequencing, Infection, Sequencing

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gku1387

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

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

    Techniques: Transfection, Western Blot