Structured Review

Thermo Fisher mnase
Digestion of different regions of the rDNA units with <t>MNase.</t> (A) Isolated 1- to 16-h embryonic nuclei were digested with MNase (0.5 U/μl) at <t>24°C</t> for 0, 1, or 4 min (lanes 2 to 4 of each panel). Purified (protein-free) genomic DNA was
Mnase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mnase/product/Thermo Fisher
Average 96 stars, based on 21 article reviews
Price from $9.99 to $1999.99
mnase - by Bioz Stars, 2020-07
96/100 stars

Images

1) Product Images from "Chromatin Structure and Transcription of the R1- and R2-Inserted rRNA Genes of Drosophila melanogaster ▿"

Article Title: Chromatin Structure and Transcription of the R1- and R2-Inserted rRNA Genes of Drosophila melanogaster ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01409-06

Digestion of different regions of the rDNA units with MNase. (A) Isolated 1- to 16-h embryonic nuclei were digested with MNase (0.5 U/μl) at 24°C for 0, 1, or 4 min (lanes 2 to 4 of each panel). Purified (protein-free) genomic DNA was
Figure Legend Snippet: Digestion of different regions of the rDNA units with MNase. (A) Isolated 1- to 16-h embryonic nuclei were digested with MNase (0.5 U/μl) at 24°C for 0, 1, or 4 min (lanes 2 to 4 of each panel). Purified (protein-free) genomic DNA was

Techniques Used: Isolation, Purification

2) Product Images from "The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells"

Article Title: The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

Journal: eLife

doi: 10.7554/eLife.03573

DKC1-associated RNAs in recombinant DKC1 complexes are resistant to extensive MNase digestion. RNAs co-purified from mock and MNase-treated recombinant DKC1 complexes in Figure 4—figure supplement 1 are 5′ end radiolabeled and separated on a 6% urea-polyacrylamide gel as described in Figure 2—figure supplement 1 . Note that the prominently labeled 130–140 nucleotide (nt)-long RNA clusters are resistant to complete nuclease digestion. It appears that these RNAs are cut on average once by MNase to generate two new smaller clusters (80–90 nt and 30–55 nt) that remain stably associated with the DKC1 complexes. Increasing the amount of MNase and/or nuclease digestion time did not change the patterns or disrupt the integrity of the protein complexes (data not shown). DOI: http://dx.doi.org/10.7554/eLife.03573.010
Figure Legend Snippet: DKC1-associated RNAs in recombinant DKC1 complexes are resistant to extensive MNase digestion. RNAs co-purified from mock and MNase-treated recombinant DKC1 complexes in Figure 4—figure supplement 1 are 5′ end radiolabeled and separated on a 6% urea-polyacrylamide gel as described in Figure 2—figure supplement 1 . Note that the prominently labeled 130–140 nucleotide (nt)-long RNA clusters are resistant to complete nuclease digestion. It appears that these RNAs are cut on average once by MNase to generate two new smaller clusters (80–90 nt and 30–55 nt) that remain stably associated with the DKC1 complexes. Increasing the amount of MNase and/or nuclease digestion time did not change the patterns or disrupt the integrity of the protein complexes (data not shown). DOI: http://dx.doi.org/10.7554/eLife.03573.010

Techniques Used: Recombinant, Purification, Labeling, Stable Transfection

Micrococcal nuclease (MNase)-treated recombinant DKC1 complexes remain structurally intact. Recombinant wild-type (WT) and various mutant DKC1 complexes are mock treated (−) or digested extensively with MNase (+), and washed extensively to remove any dissociated RNAs prior to FLAG peptide elution. Eluted protein complexes are analyzed by Coomassie Blue staining. DOI: http://dx.doi.org/10.7554/eLife.03573.009
Figure Legend Snippet: Micrococcal nuclease (MNase)-treated recombinant DKC1 complexes remain structurally intact. Recombinant wild-type (WT) and various mutant DKC1 complexes are mock treated (−) or digested extensively with MNase (+), and washed extensively to remove any dissociated RNAs prior to FLAG peptide elution. Eluted protein complexes are analyzed by Coomassie Blue staining. DOI: http://dx.doi.org/10.7554/eLife.03573.009

Techniques Used: Recombinant, Mutagenesis, Staining

MNase digestion moderately increases DKC1 coactivator activity. Mock (−) or MNase-treated (+) WT and Nop10 R34W DKC1 complexes are assayed in in vitro transcription reactions (over a fourfold concentration range) supplemented with OCT4, SOX2, recombinant XPC complex and SCC-B. DOI: http://dx.doi.org/10.7554/eLife.03573.011
Figure Legend Snippet: MNase digestion moderately increases DKC1 coactivator activity. Mock (−) or MNase-treated (+) WT and Nop10 R34W DKC1 complexes are assayed in in vitro transcription reactions (over a fourfold concentration range) supplemented with OCT4, SOX2, recombinant XPC complex and SCC-B. DOI: http://dx.doi.org/10.7554/eLife.03573.011

Techniques Used: Activity Assay, In Vitro, Concentration Assay, Recombinant

3) Product Images from "Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans"

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

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1000639

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

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

4) Product Images from "Caspase-dependent cell death-associated release of nucleosome and damage-associated molecular patterns"

Article Title: Caspase-dependent cell death-associated release of nucleosome and damage-associated molecular patterns

Journal: Cell Death & Disease

doi: 10.1038/cddis.2014.450

Release of nucleosomes and DAMPs from amino-acid-depleted HeLa cells. ( A ) An inverted microscopic image of HeLa cells in the condition of amino-acid depletion. Arrows designate dying HeLa cells. ( B ) Genomic sequences of glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), Fas, cytochrome oxidase subunit 1 ( Co1 ) and ATP synthase subunit 6 ( ATP6 ) were PCR amplified from extracellularly released DNA, genomic or mitochondrial DNAs. ( C ) Inverted and fluorescent microscopic images were taken from amino-acid-deprived HeLa cells in the presence of SYTOX, a membrane-impermeable DNA dye. HeLa cells deprived of amino acids for 15 h were fluorescence stained with histone H1 or IL6 antibodies ( D ), histone H2A, H2B, H3 or H4 antibodies ( E ) or HMGB1, Hsp90 or ERp57 antibodies ( F ) in combination with histone H1 antibody and 4',6-diamidino-2-phenylindole (DAPI). ( G ) Amino-acid-deprived HeLa cells were stained with SYTOX to determine viability, fixed and stained with DAPI and histone H1 antibodies. ( H ) Amino-acid-deprived HeLa cells were untreated or treated with MNase (500 mU/ml) for 10 min. Released DNA was quantitated at the indicated times. Data from triplicate samples are presented as mean±S.D. ( I ) Conditioned media from amino-acid-deprived HeLa cells treated or untreated with MNase were western blotted with histone H1, 2B, H3, H4, IL6, ERp57, HMGB1 or Hsp90 antibodies. ( J ) Images captured every hour from live imaging of amino-acid-deprived HeLa cells with SYTOX (green) and DRAQ5, membrane-permeable DNA dye (red). ( K ) SYTOX fluorescent intensities were measured from circularized areas of live imaging of amino-acid-deprived cells in 5-min intervals. ( L ) TEM images of control cells ( L a) and amino-acid-deprived HeLa cells ( L b– L d). ( M ) Amino-acid-deprived HeLa cells were fluorescence stained with lamin and nuclear pore antibodies, or lamin antibody and wheat germ agglutinin (WGE)
Figure Legend Snippet: Release of nucleosomes and DAMPs from amino-acid-depleted HeLa cells. ( A ) An inverted microscopic image of HeLa cells in the condition of amino-acid depletion. Arrows designate dying HeLa cells. ( B ) Genomic sequences of glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), Fas, cytochrome oxidase subunit 1 ( Co1 ) and ATP synthase subunit 6 ( ATP6 ) were PCR amplified from extracellularly released DNA, genomic or mitochondrial DNAs. ( C ) Inverted and fluorescent microscopic images were taken from amino-acid-deprived HeLa cells in the presence of SYTOX, a membrane-impermeable DNA dye. HeLa cells deprived of amino acids for 15 h were fluorescence stained with histone H1 or IL6 antibodies ( D ), histone H2A, H2B, H3 or H4 antibodies ( E ) or HMGB1, Hsp90 or ERp57 antibodies ( F ) in combination with histone H1 antibody and 4',6-diamidino-2-phenylindole (DAPI). ( G ) Amino-acid-deprived HeLa cells were stained with SYTOX to determine viability, fixed and stained with DAPI and histone H1 antibodies. ( H ) Amino-acid-deprived HeLa cells were untreated or treated with MNase (500 mU/ml) for 10 min. Released DNA was quantitated at the indicated times. Data from triplicate samples are presented as mean±S.D. ( I ) Conditioned media from amino-acid-deprived HeLa cells treated or untreated with MNase were western blotted with histone H1, 2B, H3, H4, IL6, ERp57, HMGB1 or Hsp90 antibodies. ( J ) Images captured every hour from live imaging of amino-acid-deprived HeLa cells with SYTOX (green) and DRAQ5, membrane-permeable DNA dye (red). ( K ) SYTOX fluorescent intensities were measured from circularized areas of live imaging of amino-acid-deprived cells in 5-min intervals. ( L ) TEM images of control cells ( L a) and amino-acid-deprived HeLa cells ( L b– L d). ( M ) Amino-acid-deprived HeLa cells were fluorescence stained with lamin and nuclear pore antibodies, or lamin antibody and wheat germ agglutinin (WGE)

Techniques Used: Genomic Sequencing, Polymerase Chain Reaction, Amplification, Fluorescence, Staining, Western Blot, Imaging, Transmission Electron Microscopy

5) Product Images from "CHD1 remodelers regulate nucleosome spacing in vitro and align nucleosomal arrays over gene coding regions in S. pombe"

Article Title: CHD1 remodelers regulate nucleosome spacing in vitro and align nucleosomal arrays over gene coding regions in S. pombe

Journal: The EMBO Journal

doi: 10.1038/emboj.2012.289

Hrp1 and Hrp3 efficiently space nucleosomes in vitro in an ATP-dependent manner. Phage λ DNA salt dialysis chromatin was incubated with Hrp1, Hrp3 and ATP as indicated above the lanes and digested with MNase. Wedges on top of the lanes correspond to increasing concentrations of 1, 2 and 4 U/ml. ‘M' denotes 100 bp marker (New England Biolabs).
Figure Legend Snippet: Hrp1 and Hrp3 efficiently space nucleosomes in vitro in an ATP-dependent manner. Phage λ DNA salt dialysis chromatin was incubated with Hrp1, Hrp3 and ATP as indicated above the lanes and digested with MNase. Wedges on top of the lanes correspond to increasing concentrations of 1, 2 and 4 U/ml. ‘M' denotes 100 bp marker (New England Biolabs).

Techniques Used: In Vitro, Incubation, Marker

Related Articles

Centrifugation:

Article Title: Gene silencing induced by oxidative DNA base damage: association with local decrease of histone H4 acetylation in the promoter region
Article Snippet: .. Preparation of soluble chromatin and equilibrium centrifugation 25 OD260 units of nuclei preparations were supplemented with 10 mM CaCl2 and digested with micrococcal nuclease (Fermentas, St. Leon-Rot, Germany). ..

Electrophoresis:

Article Title: Glycyl-tRNA synthetase specifically binds to the poliovirus IRES to activate translation initiation
Article Snippet: .. Finally, the beads were resuspended in 80 µl of wash buffer B supplemented with 2 mM CaCl2 and 300 U of micrococcal nuclease (Fermentas) and incubated at 37°C for 30 min. Supernatants containing RNA binding proteins were loaded on a SDS–PAGE and the gels stained with Coumassie or silver after electrophoresis. .. In vitro transcription To synthesize monocistronic uncapped RNAs bearing the 5′-UTR (wt or mutated) of PV RNA, the Fluc coding sequence, the vector 3′-UTR and polyA, the PCR products were prepared using the forward primer P9 bearing the T7 promoter sequence and the reverse primer P10.

Concentration Assay:

Article Title: Linker histones are fine-scale chromatin architects modulating developmental decisions in Arabidopsis
Article Snippet: .. Digestion was performed by incubating the nuclei suspended in DB buffer (16 mM Tris-HCl pH = 7.6, 50 mM NaCl, 2.5 mM CaCl2 , 0.01 mM PMSF, 1× Complete EDTA-free Protease Inhibitors (Roche)) with 1.5 μL (final concentration 0.3 U/μL) of micrococcal nuclease (Thermo Fisher), and 2 μL (final concentration 0.2 U/μL) of RNase A (Thermo Fisher) at 8 °C for 90 min with gentle mixing. .. The reaction was stopped by adding an equal volume of 2× Lysis buffer with EDTA (100 mM Tris-HCl pH = 8, 200 mM NaCl, 50 mM EDTA, 1% SDS).

Incubation:

Article Title: Glycyl-tRNA synthetase specifically binds to the poliovirus IRES to activate translation initiation
Article Snippet: .. Finally, the beads were resuspended in 80 µl of wash buffer B supplemented with 2 mM CaCl2 and 300 U of micrococcal nuclease (Fermentas) and incubated at 37°C for 30 min. Supernatants containing RNA binding proteins were loaded on a SDS–PAGE and the gels stained with Coumassie or silver after electrophoresis. .. In vitro transcription To synthesize monocistronic uncapped RNAs bearing the 5′-UTR (wt or mutated) of PV RNA, the Fluc coding sequence, the vector 3′-UTR and polyA, the PCR products were prepared using the forward primer P9 bearing the T7 promoter sequence and the reverse primer P10.

Article Title: Association of Bovine Papillomavirus E2 Protein with Nuclear Structures In Vivo
Article Snippet: .. Nuclei suspension was incubated with 30 U of micrococcal nuclease (Fermentas) at room temperature during the indicated time. ..

Sonication:

Article Title: PARP-1–dependent recruitment of cold-inducible RNA-binding protein promotes double-strand break repair and genome stability
Article Snippet: .. Cells were lysed, and the cross-linked nucleus lysates were digested with micrococcal nuclease and then sonicated with a Model 100 Sonic Dismembrator (Thermo Fisher Scientific) to yield genomic DNA fragments between 150 and 900 bp. .. The digested chromatin (5 μg) was immunoprecipitated with indicated primary antibody overnight at 4 °C.

Staining:

Article Title: Glycyl-tRNA synthetase specifically binds to the poliovirus IRES to activate translation initiation
Article Snippet: .. Finally, the beads were resuspended in 80 µl of wash buffer B supplemented with 2 mM CaCl2 and 300 U of micrococcal nuclease (Fermentas) and incubated at 37°C for 30 min. Supernatants containing RNA binding proteins were loaded on a SDS–PAGE and the gels stained with Coumassie or silver after electrophoresis. .. In vitro transcription To synthesize monocistronic uncapped RNAs bearing the 5′-UTR (wt or mutated) of PV RNA, the Fluc coding sequence, the vector 3′-UTR and polyA, the PCR products were prepared using the forward primer P9 bearing the T7 promoter sequence and the reverse primer P10.

RNA Binding Assay:

Article Title: Glycyl-tRNA synthetase specifically binds to the poliovirus IRES to activate translation initiation
Article Snippet: .. Finally, the beads were resuspended in 80 µl of wash buffer B supplemented with 2 mM CaCl2 and 300 U of micrococcal nuclease (Fermentas) and incubated at 37°C for 30 min. Supernatants containing RNA binding proteins were loaded on a SDS–PAGE and the gels stained with Coumassie or silver after electrophoresis. .. In vitro transcription To synthesize monocistronic uncapped RNAs bearing the 5′-UTR (wt or mutated) of PV RNA, the Fluc coding sequence, the vector 3′-UTR and polyA, the PCR products were prepared using the forward primer P9 bearing the T7 promoter sequence and the reverse primer P10.

SDS Page:

Article Title: Glycyl-tRNA synthetase specifically binds to the poliovirus IRES to activate translation initiation
Article Snippet: .. Finally, the beads were resuspended in 80 µl of wash buffer B supplemented with 2 mM CaCl2 and 300 U of micrococcal nuclease (Fermentas) and incubated at 37°C for 30 min. Supernatants containing RNA binding proteins were loaded on a SDS–PAGE and the gels stained with Coumassie or silver after electrophoresis. .. In vitro transcription To synthesize monocistronic uncapped RNAs bearing the 5′-UTR (wt or mutated) of PV RNA, the Fluc coding sequence, the vector 3′-UTR and polyA, the PCR products were prepared using the forward primer P9 bearing the T7 promoter sequence and the reverse primer P10.

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    Thermo Fisher mnase
    <t>DKC1-associated</t> RNAs in recombinant DKC1 complexes are resistant to extensive <t>MNase</t> digestion. RNAs co-purified from mock and MNase-treated recombinant DKC1 complexes in Figure 4—figure supplement 1 are 5′ end radiolabeled and separated on a 6% urea-polyacrylamide gel as described in Figure 2—figure supplement 1 . Note that the prominently labeled 130–140 nucleotide (nt)-long RNA clusters are resistant to complete nuclease digestion. It appears that these RNAs are cut on average once by MNase to generate two new smaller clusters (80–90 nt and 30–55 nt) that remain stably associated with the DKC1 complexes. Increasing the amount of MNase and/or nuclease digestion time did not change the patterns or disrupt the integrity of the protein complexes (data not shown). DOI: http://dx.doi.org/10.7554/eLife.03573.010
    Mnase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mnase/product/Thermo Fisher
    Average 92 stars, based on 21 article reviews
    Price from $9.99 to $1999.99
    mnase - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

    93
    Thermo Fisher purification gst mnase
    MapR and RHΔ CUT RUN Signals Are Enriched at Similar Regions Genome-wide (A) Schematic of RHΔC R using FLAG M2 antibody (left) and MapR using <t>GST-RHΔ-MNase</t> (right) in HEK293. (B) Enriched regions identified by RHΔC R and R-ChIP in HEK293. GRO-seq and H3K4me3 tracks indicate active gene transcription. (C) Venn diagram of gene-level overlap between RHΔC R and R-ChIP. Total number of unique genes with an R-loop at the promoter region (−2kb/+2kb from the TSS) and their overlap are shown. p
    Purification Gst Mnase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/purification gst mnase/product/Thermo Fisher
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    purification gst mnase - by Bioz Stars, 2020-07
    93/100 stars
      Buy from Supplier

    Image Search Results


    DKC1-associated RNAs in recombinant DKC1 complexes are resistant to extensive MNase digestion. RNAs co-purified from mock and MNase-treated recombinant DKC1 complexes in Figure 4—figure supplement 1 are 5′ end radiolabeled and separated on a 6% urea-polyacrylamide gel as described in Figure 2—figure supplement 1 . Note that the prominently labeled 130–140 nucleotide (nt)-long RNA clusters are resistant to complete nuclease digestion. It appears that these RNAs are cut on average once by MNase to generate two new smaller clusters (80–90 nt and 30–55 nt) that remain stably associated with the DKC1 complexes. Increasing the amount of MNase and/or nuclease digestion time did not change the patterns or disrupt the integrity of the protein complexes (data not shown). DOI: http://dx.doi.org/10.7554/eLife.03573.010

    Journal: eLife

    Article Title: The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

    doi: 10.7554/eLife.03573

    Figure Lengend Snippet: DKC1-associated RNAs in recombinant DKC1 complexes are resistant to extensive MNase digestion. RNAs co-purified from mock and MNase-treated recombinant DKC1 complexes in Figure 4—figure supplement 1 are 5′ end radiolabeled and separated on a 6% urea-polyacrylamide gel as described in Figure 2—figure supplement 1 . Note that the prominently labeled 130–140 nucleotide (nt)-long RNA clusters are resistant to complete nuclease digestion. It appears that these RNAs are cut on average once by MNase to generate two new smaller clusters (80–90 nt and 30–55 nt) that remain stably associated with the DKC1 complexes. Increasing the amount of MNase and/or nuclease digestion time did not change the patterns or disrupt the integrity of the protein complexes (data not shown). DOI: http://dx.doi.org/10.7554/eLife.03573.010

    Article Snippet: Bound DKC1 complexes were treated with 300 U of MNase (Thermo Scientific, Waltham, MA) or buffer at room temperature and nutated for 1 hr.

    Techniques: Recombinant, Purification, Labeling, Stable Transfection

    Micrococcal nuclease (MNase)-treated recombinant DKC1 complexes remain structurally intact. Recombinant wild-type (WT) and various mutant DKC1 complexes are mock treated (−) or digested extensively with MNase (+), and washed extensively to remove any dissociated RNAs prior to FLAG peptide elution. Eluted protein complexes are analyzed by Coomassie Blue staining. DOI: http://dx.doi.org/10.7554/eLife.03573.009

    Journal: eLife

    Article Title: The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

    doi: 10.7554/eLife.03573

    Figure Lengend Snippet: Micrococcal nuclease (MNase)-treated recombinant DKC1 complexes remain structurally intact. Recombinant wild-type (WT) and various mutant DKC1 complexes are mock treated (−) or digested extensively with MNase (+), and washed extensively to remove any dissociated RNAs prior to FLAG peptide elution. Eluted protein complexes are analyzed by Coomassie Blue staining. DOI: http://dx.doi.org/10.7554/eLife.03573.009

    Article Snippet: Bound DKC1 complexes were treated with 300 U of MNase (Thermo Scientific, Waltham, MA) or buffer at room temperature and nutated for 1 hr.

    Techniques: Recombinant, Mutagenesis, Staining

    MNase digestion moderately increases DKC1 coactivator activity. Mock (−) or MNase-treated (+) WT and Nop10 R34W DKC1 complexes are assayed in in vitro transcription reactions (over a fourfold concentration range) supplemented with OCT4, SOX2, recombinant XPC complex and SCC-B. DOI: http://dx.doi.org/10.7554/eLife.03573.011

    Journal: eLife

    Article Title: The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

    doi: 10.7554/eLife.03573

    Figure Lengend Snippet: MNase digestion moderately increases DKC1 coactivator activity. Mock (−) or MNase-treated (+) WT and Nop10 R34W DKC1 complexes are assayed in in vitro transcription reactions (over a fourfold concentration range) supplemented with OCT4, SOX2, recombinant XPC complex and SCC-B. DOI: http://dx.doi.org/10.7554/eLife.03573.011

    Article Snippet: Bound DKC1 complexes were treated with 300 U of MNase (Thermo Scientific, Waltham, MA) or buffer at room temperature and nutated for 1 hr.

    Techniques: Activity Assay, In Vitro, Concentration Assay, Recombinant

    Release of nucleosomes and DAMPs from amino-acid-depleted HeLa cells. ( A ) An inverted microscopic image of HeLa cells in the condition of amino-acid depletion. Arrows designate dying HeLa cells. ( B ) Genomic sequences of glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), Fas, cytochrome oxidase subunit 1 ( Co1 ) and ATP synthase subunit 6 ( ATP6 ) were PCR amplified from extracellularly released DNA, genomic or mitochondrial DNAs. ( C ) Inverted and fluorescent microscopic images were taken from amino-acid-deprived HeLa cells in the presence of SYTOX, a membrane-impermeable DNA dye. HeLa cells deprived of amino acids for 15 h were fluorescence stained with histone H1 or IL6 antibodies ( D ), histone H2A, H2B, H3 or H4 antibodies ( E ) or HMGB1, Hsp90 or ERp57 antibodies ( F ) in combination with histone H1 antibody and 4',6-diamidino-2-phenylindole (DAPI). ( G ) Amino-acid-deprived HeLa cells were stained with SYTOX to determine viability, fixed and stained with DAPI and histone H1 antibodies. ( H ) Amino-acid-deprived HeLa cells were untreated or treated with MNase (500 mU/ml) for 10 min. Released DNA was quantitated at the indicated times. Data from triplicate samples are presented as mean±S.D. ( I ) Conditioned media from amino-acid-deprived HeLa cells treated or untreated with MNase were western blotted with histone H1, 2B, H3, H4, IL6, ERp57, HMGB1 or Hsp90 antibodies. ( J ) Images captured every hour from live imaging of amino-acid-deprived HeLa cells with SYTOX (green) and DRAQ5, membrane-permeable DNA dye (red). ( K ) SYTOX fluorescent intensities were measured from circularized areas of live imaging of amino-acid-deprived cells in 5-min intervals. ( L ) TEM images of control cells ( L a) and amino-acid-deprived HeLa cells ( L b– L d). ( M ) Amino-acid-deprived HeLa cells were fluorescence stained with lamin and nuclear pore antibodies, or lamin antibody and wheat germ agglutinin (WGE)

    Journal: Cell Death & Disease

    Article Title: Caspase-dependent cell death-associated release of nucleosome and damage-associated molecular patterns

    doi: 10.1038/cddis.2014.450

    Figure Lengend Snippet: Release of nucleosomes and DAMPs from amino-acid-depleted HeLa cells. ( A ) An inverted microscopic image of HeLa cells in the condition of amino-acid depletion. Arrows designate dying HeLa cells. ( B ) Genomic sequences of glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), Fas, cytochrome oxidase subunit 1 ( Co1 ) and ATP synthase subunit 6 ( ATP6 ) were PCR amplified from extracellularly released DNA, genomic or mitochondrial DNAs. ( C ) Inverted and fluorescent microscopic images were taken from amino-acid-deprived HeLa cells in the presence of SYTOX, a membrane-impermeable DNA dye. HeLa cells deprived of amino acids for 15 h were fluorescence stained with histone H1 or IL6 antibodies ( D ), histone H2A, H2B, H3 or H4 antibodies ( E ) or HMGB1, Hsp90 or ERp57 antibodies ( F ) in combination with histone H1 antibody and 4',6-diamidino-2-phenylindole (DAPI). ( G ) Amino-acid-deprived HeLa cells were stained with SYTOX to determine viability, fixed and stained with DAPI and histone H1 antibodies. ( H ) Amino-acid-deprived HeLa cells were untreated or treated with MNase (500 mU/ml) for 10 min. Released DNA was quantitated at the indicated times. Data from triplicate samples are presented as mean±S.D. ( I ) Conditioned media from amino-acid-deprived HeLa cells treated or untreated with MNase were western blotted with histone H1, 2B, H3, H4, IL6, ERp57, HMGB1 or Hsp90 antibodies. ( J ) Images captured every hour from live imaging of amino-acid-deprived HeLa cells with SYTOX (green) and DRAQ5, membrane-permeable DNA dye (red). ( K ) SYTOX fluorescent intensities were measured from circularized areas of live imaging of amino-acid-deprived cells in 5-min intervals. ( L ) TEM images of control cells ( L a) and amino-acid-deprived HeLa cells ( L b– L d). ( M ) Amino-acid-deprived HeLa cells were fluorescence stained with lamin and nuclear pore antibodies, or lamin antibody and wheat germ agglutinin (WGE)

    Article Snippet: Released DNA from dying cells were digested with 500 mU/ml MNase (Thermo Scientific) for 5 min. Nuclease activity was stopped with 5 mM EDTA and the culture supernatants were collected and stored at −20 °C until quantification.

    Techniques: Genomic Sequencing, Polymerase Chain Reaction, Amplification, Fluorescence, Staining, Western Blot, Imaging, Transmission Electron Microscopy

    MapR and RHΔ CUT RUN Signals Are Enriched at Similar Regions Genome-wide (A) Schematic of RHΔC R using FLAG M2 antibody (left) and MapR using GST-RHΔ-MNase (right) in HEK293. (B) Enriched regions identified by RHΔC R and R-ChIP in HEK293. GRO-seq and H3K4me3 tracks indicate active gene transcription. (C) Venn diagram of gene-level overlap between RHΔC R and R-ChIP. Total number of unique genes with an R-loop at the promoter region (−2kb/+2kb from the TSS) and their overlap are shown. p

    Journal: Cell reports

    Article Title: Mapping Native R-Loops Genome-wide Using a Targeted Nuclease Approach

    doi: 10.1016/j.celrep.2019.09.052

    Figure Lengend Snippet: MapR and RHΔ CUT RUN Signals Are Enriched at Similar Regions Genome-wide (A) Schematic of RHΔC R using FLAG M2 antibody (left) and MapR using GST-RHΔ-MNase (right) in HEK293. (B) Enriched regions identified by RHΔC R and R-ChIP in HEK293. GRO-seq and H3K4me3 tracks indicate active gene transcription. (C) Venn diagram of gene-level overlap between RHΔC R and R-ChIP. Total number of unique genes with an R-loop at the promoter region (−2kb/+2kb from the TSS) and their overlap are shown. p

    Article Snippet: Protein Expression and Purification GST-MNase and GST-RHΔMNase were cloned into pGEX plasmid and transformed into BL21 (DE3) (ThermoFisher C601003) for expression.

    Techniques: Genome Wide, Chromatin Immunoprecipitation

    MapR, a Native and Antibody-Independent R-Loop Detection Strategy R-loop recognition and recovery by MapR. Step 1: cells are immobilized on concanavalin A beads and permeabilized. Step 2: equimolar amounts of a catalytic deficient mutant of RNase H fused to micrococcal nuclease (GST-RHΔ-MNase) or GST-MNase is added to immobilized cells. Step 3: the RHΔ module recognizes and binds R-loops on chromatin. Step 4: controlled activation of the MNase moiety by addition of calcium results in cleavage of DNA fragments in proximity to R-loops. Step 5: Released R-loops diffuse out of the cell; the DNA is recovered and sequenced.

    Journal: Cell reports

    Article Title: Mapping Native R-Loops Genome-wide Using a Targeted Nuclease Approach

    doi: 10.1016/j.celrep.2019.09.052

    Figure Lengend Snippet: MapR, a Native and Antibody-Independent R-Loop Detection Strategy R-loop recognition and recovery by MapR. Step 1: cells are immobilized on concanavalin A beads and permeabilized. Step 2: equimolar amounts of a catalytic deficient mutant of RNase H fused to micrococcal nuclease (GST-RHΔ-MNase) or GST-MNase is added to immobilized cells. Step 3: the RHΔ module recognizes and binds R-loops on chromatin. Step 4: controlled activation of the MNase moiety by addition of calcium results in cleavage of DNA fragments in proximity to R-loops. Step 5: Released R-loops diffuse out of the cell; the DNA is recovered and sequenced.

    Article Snippet: Protein Expression and Purification GST-MNase and GST-RHΔMNase were cloned into pGEX plasmid and transformed into BL21 (DE3) (ThermoFisher C601003) for expression.

    Techniques: Mutagenesis, Activation Assay