mnase  (Worthington Biochemical)


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    Structured Review

    Worthington Biochemical mnase
    <t>Bbd-containing</t> nucleosomal arrays repress basal transcription. ( A ) In all, 2.1 μg of assembled Bbd-, mouse- and Drosophila -nucleosomal arrays was digested with 0.12 U <t>MNase.</t> Aliquots of 0.5 μg were removed after 1, 2, 4 and 8 min, deproteinized and analyzed on 1.2% agarose gels. ( B ) In vitro transcription reactions for naked DNA, Bbd-nucleosomal arrays and mouse-nucleosomal arrays are shown. Tax/CREB and p300 were added as indicated above the lanes. Recovery standard and transcript are indicated on the left. ( C ) Results from three transcription assays (from three independent chromatin assembly reactions) were quantified by ImageQuant 5.1 and normalized compared to the recovery standard. Transcripts from mouse and Bbd-nucleosomal arrays are shown by white and gray bars, respectively.
    Mnase, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 96/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mnase - by Bioz Stars, 2020-01
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    Images

    1) Product Images from "Nucleosomes containing the histone variant H2A.Bbd organize only 118 base pairs of DNA"

    Article Title: Nucleosomes containing the histone variant H2A.Bbd organize only 118 base pairs of DNA

    Journal: The EMBO Journal

    doi: 10.1038/sj.emboj.7600316

    Bbd-containing nucleosomal arrays repress basal transcription. ( A ) In all, 2.1 μg of assembled Bbd-, mouse- and Drosophila -nucleosomal arrays was digested with 0.12 U MNase. Aliquots of 0.5 μg were removed after 1, 2, 4 and 8 min, deproteinized and analyzed on 1.2% agarose gels. ( B ) In vitro transcription reactions for naked DNA, Bbd-nucleosomal arrays and mouse-nucleosomal arrays are shown. Tax/CREB and p300 were added as indicated above the lanes. Recovery standard and transcript are indicated on the left. ( C ) Results from three transcription assays (from three independent chromatin assembly reactions) were quantified by ImageQuant 5.1 and normalized compared to the recovery standard. Transcripts from mouse and Bbd-nucleosomal arrays are shown by white and gray bars, respectively.
    Figure Legend Snippet: Bbd-containing nucleosomal arrays repress basal transcription. ( A ) In all, 2.1 μg of assembled Bbd-, mouse- and Drosophila -nucleosomal arrays was digested with 0.12 U MNase. Aliquots of 0.5 μg were removed after 1, 2, 4 and 8 min, deproteinized and analyzed on 1.2% agarose gels. ( B ) In vitro transcription reactions for naked DNA, Bbd-nucleosomal arrays and mouse-nucleosomal arrays are shown. Tax/CREB and p300 were added as indicated above the lanes. Recovery standard and transcript are indicated on the left. ( C ) Results from three transcription assays (from three independent chromatin assembly reactions) were quantified by ImageQuant 5.1 and normalized compared to the recovery standard. Transcripts from mouse and Bbd-nucleosomal arrays are shown by white and gray bars, respectively.

    Techniques Used: In Vitro

    Bbd-NCPs are less resistant against MNase digestion. Identical amounts (2 μg) of mm -146-NCP ( A ) and Bbd-146-NCP ( B ) were digested with increasing amounts of MNase (0, 0.05, 0.1, 0.2, 0.4 U) for 1.5 min. Deproteinized samples were analyzed by 10% PAGE, stained with ethidium bromide. For time courses, 10 μg of mm -196-NCP ( C ) and Bbd-196-NCP ( D ) was incubated with the same amount of MNase (0.15 U) for the indicated times. In all, 2 μg of sample was removed at 0, 2, 4, 8 and 16 min, and the deproteinized samples were analyzed by 10% PAGE. The 146 bp 5sDNA (146), 196 bp 5sDNA (196) and mixtures of 146 and 73 bp α-satellite DNA (146+73) were loaded as controls, as indicated. DNA size markers are given.
    Figure Legend Snippet: Bbd-NCPs are less resistant against MNase digestion. Identical amounts (2 μg) of mm -146-NCP ( A ) and Bbd-146-NCP ( B ) were digested with increasing amounts of MNase (0, 0.05, 0.1, 0.2, 0.4 U) for 1.5 min. Deproteinized samples were analyzed by 10% PAGE, stained with ethidium bromide. For time courses, 10 μg of mm -196-NCP ( C ) and Bbd-196-NCP ( D ) was incubated with the same amount of MNase (0.15 U) for the indicated times. In all, 2 μg of sample was removed at 0, 2, 4, 8 and 16 min, and the deproteinized samples were analyzed by 10% PAGE. The 146 bp 5sDNA (146), 196 bp 5sDNA (196) and mixtures of 146 and 73 bp α-satellite DNA (146+73) were loaded as controls, as indicated. DNA size markers are given.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Staining, Incubation

    Xla H2A 106 -NCP and Xla H2A 112 -NCP behave like wild-type mm- NCP. ( A ) Salt gradient reconstituted Bbd-NCP (lane 1), Xla -NCP (lanes 6 and 7), Xla H2A 106 -NCP (106NCP, lanes 2 and 3) and Xla H2A 112 -NCP (112NCP, lanes 4 and 5), before (−) and after (+) a 1 h incubation at 37°C, were analyzed by 5% native PAGE, followed by staining with Coomassie blue. ( B–E ) Micrococcal digestion of mutant nucleosomes: 10 μg of Xla -NCP (B), Bbd-NCP (C), Xla H2A 112 -NCP (D) and Xla H2A 106 -NCP (E) were digested with 0.15 U MNase. Aliquots of 2 μg were removed after 0, 2, 4, 8 and 16 min. PAGE (10%) was used to check the deproteinized DNA fragments. 5SDNA (146 bp) (146) was loaded as indicated (D and E, lane 8).
    Figure Legend Snippet: Xla H2A 106 -NCP and Xla H2A 112 -NCP behave like wild-type mm- NCP. ( A ) Salt gradient reconstituted Bbd-NCP (lane 1), Xla -NCP (lanes 6 and 7), Xla H2A 106 -NCP (106NCP, lanes 2 and 3) and Xla H2A 112 -NCP (112NCP, lanes 4 and 5), before (−) and after (+) a 1 h incubation at 37°C, were analyzed by 5% native PAGE, followed by staining with Coomassie blue. ( B–E ) Micrococcal digestion of mutant nucleosomes: 10 μg of Xla -NCP (B), Bbd-NCP (C), Xla H2A 112 -NCP (D) and Xla H2A 106 -NCP (E) were digested with 0.15 U MNase. Aliquots of 2 μg were removed after 0, 2, 4, 8 and 16 min. PAGE (10%) was used to check the deproteinized DNA fragments. 5SDNA (146 bp) (146) was loaded as indicated (D and E, lane 8).

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

    2) Product Images from "Zelda overcomes the high intrinsic nucleosome barrier at enhancers during Drosophila zygotic genome activation"

    Article Title: Zelda overcomes the high intrinsic nucleosome barrier at enhancers during Drosophila zygotic genome activation

    Journal: Genome Research

    doi: 10.1101/gr.192542.115

    Zld binding and changes in nucleosome occupancy correlate with early enhancer activity. Analysis of 6008 Zld-bound regions that are > 1 kb away from a TSS. In the heatmaps, normalized MNase-seq and ChIP-seq data for each region are aligned to the Zld summit, and 1-kb regions to each side are shown. ( A ) All 6008 non-TSS Zld-bound regions ranked by Zld summit reads from high to low show that Zld binding strongly correlates with Zld motifs and that the higher the Zld peak ranks, the more frequently early enhancers (E), HOT regions (HOT), and Dl peaks (Dl) overlap. The Zld peak rank also correlates with the degree of hotness, i.e., the number of TFs bound, and Dl binding strength, which are shown as degree of red in the same data. White indicates non-HOT regions. ( B – D ) The 6008 Zld-bound regions are ranked by Zld summit reads from high to low as in A ( B ); wt MNase (within 250 bp of Zld summits) from low to high ( C ), and ΔMNase (the read count difference between zld − and wt ) from high to low ( D ). The ranked data were then divided into bins of 500 regions (except for the last bin, which has 508 regions), and enrichment values for each bin are shown (blue for depletion and red for enrichment). The enrichment of Zld motifs (Zld m.) was calculated over genome background, while the enrichment of early enhancers (Early), HOT regions (HOT), and Dl peaks (Dl) was calculated over the average of the 6008 Zld-bound regions. Note that the enrichment at the top two bins is strongest when ranked by Zld summit reads, still strong when ranked by ΔMNase, and the lowest when ranked by wt MNase. Heatmaps for the top two bins (1000 regions) in each ranking is shown to the right for the following data: Zld binding, wt MNase, zld − ), and ΔMNase. Note that the nucleosome occupancy in zld − embryos resembles that of late muscle tissue, where Zld is also absent, as well as the predicted model, suggesting that in the absence of Zld, nucleosome occupancy is governed by DNA sequence features.
    Figure Legend Snippet: Zld binding and changes in nucleosome occupancy correlate with early enhancer activity. Analysis of 6008 Zld-bound regions that are > 1 kb away from a TSS. In the heatmaps, normalized MNase-seq and ChIP-seq data for each region are aligned to the Zld summit, and 1-kb regions to each side are shown. ( A ) All 6008 non-TSS Zld-bound regions ranked by Zld summit reads from high to low show that Zld binding strongly correlates with Zld motifs and that the higher the Zld peak ranks, the more frequently early enhancers (E), HOT regions (HOT), and Dl peaks (Dl) overlap. The Zld peak rank also correlates with the degree of hotness, i.e., the number of TFs bound, and Dl binding strength, which are shown as degree of red in the same data. White indicates non-HOT regions. ( B – D ) The 6008 Zld-bound regions are ranked by Zld summit reads from high to low as in A ( B ); wt MNase (within 250 bp of Zld summits) from low to high ( C ), and ΔMNase (the read count difference between zld − and wt ) from high to low ( D ). The ranked data were then divided into bins of 500 regions (except for the last bin, which has 508 regions), and enrichment values for each bin are shown (blue for depletion and red for enrichment). The enrichment of Zld motifs (Zld m.) was calculated over genome background, while the enrichment of early enhancers (Early), HOT regions (HOT), and Dl peaks (Dl) was calculated over the average of the 6008 Zld-bound regions. Note that the enrichment at the top two bins is strongest when ranked by Zld summit reads, still strong when ranked by ΔMNase, and the lowest when ranked by wt MNase. Heatmaps for the top two bins (1000 regions) in each ranking is shown to the right for the following data: Zld binding, wt MNase, zld − ), and ΔMNase. Note that the nucleosome occupancy in zld − embryos resembles that of late muscle tissue, where Zld is also absent, as well as the predicted model, suggesting that in the absence of Zld, nucleosome occupancy is governed by DNA sequence features.

    Techniques Used: Binding Assay, Activity Assay, Chromatin Immunoprecipitation, Sequencing

    Loss of Dl binding in the absence of Zld is associated with increased nucleosome occupancy. Nucleosome occupancy was measured by MNase-seq (see Methods). ( A ) Metaprofiles ( wt in blue, zld − in red) of Zld-bound Dl peaks that are > 1 kb away from a TSS are shown for the three Dl-peak groups, as well as Dl peaks that do not colocalize with Zld binding as control. The normalized MNase reads were aligned at the Dl summit, and average reads within 1 kb distance are shown. ( B ) Metaprofiles of Zld-bound Dl peaks that are ≤1 kb away from a TSS are shown for Groups I and II. The normalized MNase reads were either aligned at Dl summits ( left ) or the nearby TSSs ( right ). Note that the increased nucleosome occupancy in zld − within Group I is much more pronounced on the left , arguing that the effect is directly due to Zld binding and not due to loss of transcription at these genes. ( C ) Scatter plot showing the correlation between ΔMNase ( x -axis) and the fold change in Dl binding ( y -axis) between zld − and wt embryos. Values were calculated using the reads within 125 or 250 bp of the Dl summit for Dl binding and MNase, respectively. Note the strong correlation for Group I Dl peaks (red). ( D ) Metaprofiles of the Dl-peak groups as in A ). Note that the high and broad nucleosome occupancy of Group I regions in zld − is also predicted by the model (arrow), indicating that the role of Zld may be to tackle the intrinsically strong nucleosome barrier of 4–5 nucleosomes at these places, which would then help Dl access these regions.
    Figure Legend Snippet: Loss of Dl binding in the absence of Zld is associated with increased nucleosome occupancy. Nucleosome occupancy was measured by MNase-seq (see Methods). ( A ) Metaprofiles ( wt in blue, zld − in red) of Zld-bound Dl peaks that are > 1 kb away from a TSS are shown for the three Dl-peak groups, as well as Dl peaks that do not colocalize with Zld binding as control. The normalized MNase reads were aligned at the Dl summit, and average reads within 1 kb distance are shown. ( B ) Metaprofiles of Zld-bound Dl peaks that are ≤1 kb away from a TSS are shown for Groups I and II. The normalized MNase reads were either aligned at Dl summits ( left ) or the nearby TSSs ( right ). Note that the increased nucleosome occupancy in zld − within Group I is much more pronounced on the left , arguing that the effect is directly due to Zld binding and not due to loss of transcription at these genes. ( C ) Scatter plot showing the correlation between ΔMNase ( x -axis) and the fold change in Dl binding ( y -axis) between zld − and wt embryos. Values were calculated using the reads within 125 or 250 bp of the Dl summit for Dl binding and MNase, respectively. Note the strong correlation for Group I Dl peaks (red). ( D ) Metaprofiles of the Dl-peak groups as in A ). Note that the high and broad nucleosome occupancy of Group I regions in zld − is also predicted by the model (arrow), indicating that the role of Zld may be to tackle the intrinsically strong nucleosome barrier of 4–5 nucleosomes at these places, which would then help Dl access these regions.

    Techniques Used: Binding Assay

    3) Product Images from "Tax Abolishes Histone H1 Repression of p300 Acetyltransferase Activity at the Human T-Cell Leukemia Virus Type 1 Promoter ▿"

    Article Title: Tax Abolishes Histone H1 Repression of p300 Acetyltransferase Activity at the Human T-Cell Leukemia Virus Type 1 Promoter ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.00631-06

    Histone H1 is properly incorporated into chromatin through use of a recombinant assembly system. (A) Histone H1 incorporation (1.25 molar excess to octamer) increases nucleosome repeat length from ∼170 to ∼190 bp. Chromatin was assembled on the p-306/G-less template (0.94 pmol) through use of recombinant NAP-1 and ACF assembly factors in the absence or presence of histone H1 as indicated and digested with MNase for 1, 2, 4, and 8 min. Nucleosomal DNA was resolved on a native 1.2% agarose gel and visualized by SYBR gold staining. The numbers and positions of nucleosomes for each template are labeled numerically. Base pair size markers are indicated to the right. (B) Histone H1 is stably incorporated into chromatin. Chromatin containing H1 was purified over 15% to 40% sucrose gradients. Proteins from each fraction were resolved on 15% SDS-PAGE and visualized with SYPRO ruby (top). Molecular weight markers (M) are shown on the left. Positions of H1, core histones, and the major chromatin peak fractions are also indicated. DNA from each fraction was also resolved using agarose gel electrophoresis and SYBR gold (bottom).
    Figure Legend Snippet: Histone H1 is properly incorporated into chromatin through use of a recombinant assembly system. (A) Histone H1 incorporation (1.25 molar excess to octamer) increases nucleosome repeat length from ∼170 to ∼190 bp. Chromatin was assembled on the p-306/G-less template (0.94 pmol) through use of recombinant NAP-1 and ACF assembly factors in the absence or presence of histone H1 as indicated and digested with MNase for 1, 2, 4, and 8 min. Nucleosomal DNA was resolved on a native 1.2% agarose gel and visualized by SYBR gold staining. The numbers and positions of nucleosomes for each template are labeled numerically. Base pair size markers are indicated to the right. (B) Histone H1 is stably incorporated into chromatin. Chromatin containing H1 was purified over 15% to 40% sucrose gradients. Proteins from each fraction were resolved on 15% SDS-PAGE and visualized with SYPRO ruby (top). Molecular weight markers (M) are shown on the left. Positions of H1, core histones, and the major chromatin peak fractions are also indicated. DNA from each fraction was also resolved using agarose gel electrophoresis and SYBR gold (bottom).

    Techniques Used: Recombinant, Agarose Gel Electrophoresis, Staining, Labeling, Stable Transfection, Purification, SDS Page, Molecular Weight

    4) Product Images from "Coordinated repressive chromatin-remodeling of Oct4 and Nanog genes in RA-induced differentiation of embryonic stem cells involves RIP140"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku092

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

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

    5) Product Images from "Promyelocytic leukemia protein in retinoic acid-induced chromatin remodeling of Oct4 gene promoter"

    Article Title: Promyelocytic leukemia protein in retinoic acid-induced chromatin remodeling of Oct4 gene promoter

    Journal: Stem Cells (Dayton, Ohio)

    doi: 10.1002/stem.623

    Pml silencing and RA treatment induce similar chromatin remodeling events on Oct4 PP (A) MNase mapping of the Oct4 regulatory region. Top panel shows the map of Oct4 gene promoter and enhancer region (DE, PE, PP) and four CRs. HindIII and ScaI sites and Southern blot probes (PI, PII and PIII for PP, PE and DE, respectively) are depicted. P19 stem, siPml- or RA-treated cells were subjected to MNase digestion for 5 minutes. Extracted chromatin DNA was separated on 1.5% agarose gels followed by Southern hybridization. The panels directly under the map show EtBr-stained gels and bottom panels show Southern blots probed with PI, PII or PIII. (B) Restriction enzyme accessibility of Oct4 PP region in P19 stem, siPml-treated, or RA-treated cells. “*” signs mark diagnostic bands indicative of sensitive sites. The results are summarized on the map above these blots. Restriction sites are labeled under the top map. A small black box on each map depicts the Sp1-binding site and HRE cluster on CR1. Abbreviations: DE, distal enhancer; PE, proximal enhancer; PP, proximal promoter; TIS, transcription initiation site.
    Figure Legend Snippet: Pml silencing and RA treatment induce similar chromatin remodeling events on Oct4 PP (A) MNase mapping of the Oct4 regulatory region. Top panel shows the map of Oct4 gene promoter and enhancer region (DE, PE, PP) and four CRs. HindIII and ScaI sites and Southern blot probes (PI, PII and PIII for PP, PE and DE, respectively) are depicted. P19 stem, siPml- or RA-treated cells were subjected to MNase digestion for 5 minutes. Extracted chromatin DNA was separated on 1.5% agarose gels followed by Southern hybridization. The panels directly under the map show EtBr-stained gels and bottom panels show Southern blots probed with PI, PII or PIII. (B) Restriction enzyme accessibility of Oct4 PP region in P19 stem, siPml-treated, or RA-treated cells. “*” signs mark diagnostic bands indicative of sensitive sites. The results are summarized on the map above these blots. Restriction sites are labeled under the top map. A small black box on each map depicts the Sp1-binding site and HRE cluster on CR1. Abbreviations: DE, distal enhancer; PE, proximal enhancer; PP, proximal promoter; TIS, transcription initiation site.

    Techniques Used: Southern Blot, Hybridization, Staining, Diagnostic Assay, Labeling, Binding Assay

    6) Product Images from "Subtelomeric proteins negatively regulate telomere elongation in budding yeast"

    Article Title: Subtelomeric proteins negatively regulate telomere elongation in budding yeast

    Journal: The EMBO Journal

    doi: 10.1038/sj.emboj.7600975

    Structure of the telomeric end-complex and subtelomeric chromatin in TEL1 and tel1 Δ cells. ( A ) DNA was isolated from fresh cell lysates treated with DNase I or MNase and then analyzed by Southern blot with a TG 1–3 probe. ( B ) DNA was isolated from fresh cell lysates treated with DNase I or MNase and then analyzed by Southern blot with a Y′ probe. ( C ) Analysis of the profiles of the TG 1–3 hybridizing DNA. The thick line represents the tel1 Δ samples. The thin line represents the TEL1 samples.
    Figure Legend Snippet: Structure of the telomeric end-complex and subtelomeric chromatin in TEL1 and tel1 Δ cells. ( A ) DNA was isolated from fresh cell lysates treated with DNase I or MNase and then analyzed by Southern blot with a TG 1–3 probe. ( B ) DNA was isolated from fresh cell lysates treated with DNase I or MNase and then analyzed by Southern blot with a Y′ probe. ( C ) Analysis of the profiles of the TG 1–3 hybridizing DNA. The thick line represents the tel1 Δ samples. The thin line represents the TEL1 samples.

    Techniques Used: Isolation, Southern Blot

    Related Articles

    Centrifugation:

    Article Title: Switching roles for DNA and histone methylation depend on evolutionary ages of human endogenous retroviruses
    Article Snippet: To prepare nuclei, cells were lysed in 1 mL lysis buffer (buffer N supplemented with 0.3% NP-40 substitute [Sigma]) for 10 min at 4°C, and nuclei were collected by centrifugation (500 g for 5 min at 4°C), resuspended in 1 mL of buffer N, and then sedimented through 7.5 mL sucrose cushion (10 mM HEPES pH 7.9, 30% [w/v] sucrose, 1.5 mM MgCl2 ) at 13,000 g using a Sorvall swinging bucket for 12 min at 4°C. .. To isolate single nucleosomes, the nuclei were digested with MNase (1 unit Worthington MNase per 70 μg of chromatin for 10 min at 37°C).

    Article Title: Dual inhibition of DNA and histone methyltransferases increases viral mimicry in ovarian cancer cells
    Article Snippet: To prepare nuclei, cells were lysed in 1 mL Lysis Buffer (Buffer N supplemented with 0.3% NP-40 substitute (Sigma)) for 10 min at 4°C, and nuclei were collected by centrifugation (500 × g for 5 min at 4°C), resuspended in 1 mL of Buffer N, then sedimented through 7.5 mL sucrose cushion (10 mM HEPES pH7.9, 30%(w/v) sucrose, 1.5 mM MgCl2 and centrifuged 13000 × g using Sorvall swinging bucket for 12 min at 4°C). .. To isolate single nucleosomes, the nuclei were digested with MNase (1U Worthington MNase per 70 μg of chromatin at 37°C for 10 min), the nucleosomes were then purified by hydroxyapatite chromatography, and adjusted to a concentration of 20 μg/mL with ChIP Buffer 1(25 mM Tris pH 7.5, 5 mM MgCl2, 100 mM KCl, 10% (v/v) glycerol, 0.1% (v/v) NP-40 substitute) and analyzed using 2% agrose gel.

    Article Title: Genome-wide studies reveal novel and distinct biological pathways regulated by SIN3 isoforms
    Article Snippet: These cells were disrupted by dounce homogenizer (10 times by loose pestle, 15 times by tight pestle) followed by low speed centrifugation (170 x g for 10 min). .. The pellet was resuspended in 200 μl MNase digest buffer (15 mM Tris-HCl pH 8, 60 mM KCl, 15 mM NaCl, 1 mM DTT, 0.25 M sucrose, 1 mM CaCl2 ) and subjected to micrococcal nuclease digestion by addition of 20 units of MNase (Worthington Biochemicals) for 10 min at room temperature.

    Incubation:

    Article Title: H3.3/H2A.Z double variant-containing nucleosomes mark 'nucleosome-free regions' of active promoters and other regulatory regions in the human genome
    Article Snippet: The A260 was adjusted to 1.25, and the resuspended nuclei were digested with 12 × 10-2 U/μL MNase (Worthington) for 10 min at 37°C to generate mostly mononucleosomes. .. The pellet was resuspended in lysis buffer plus 0.25 mM EDTA, incubated on ice for 15 min, and recentrifuged at 10,000 rpm for 10 min after passing four times through a 20-gauge needle followed by four passes through a 25-gauge needle.

    Article Title: Genome-wide studies reveal novel and distinct biological pathways regulated by SIN3 isoforms
    Article Snippet: The cross-linked cells were washed with 1X phosphate buffered saline and resuspended in lysis buffer (10 mM Tris-HCl pH 8, 10 mM KCl, 3 mM CaCl2 , 0.34 M sucrose, 1 mM DTT, 0.1 % Triton X-100, 0.2 mM EGTA, proteinase inhibitor (Roche)), followed by incubation on ice for 15 min. .. The pellet was resuspended in 200 μl MNase digest buffer (15 mM Tris-HCl pH 8, 60 mM KCl, 15 mM NaCl, 1 mM DTT, 0.25 M sucrose, 1 mM CaCl2 ) and subjected to micrococcal nuclease digestion by addition of 20 units of MNase (Worthington Biochemicals) for 10 min at room temperature.

    Article Title: Tax Abolishes Histone H1 Repression of p300 Acetyltransferase Activity at the Human T-Cell Leukemia Virus Type 1 Promoter ▿
    Article Snippet: .. First, 135 μl of MNase buffer (1.74 mM HEPES [pH 7.6], 121 mM KCl, 12% [vol/vol] glycerol, 2.4% [wt/vol] polyethylene glycol, 11.2 mM CaCl2 ) were added to chromatin samples and incubated at 37°C for 5 min. Digestion was initiated by addition of 0.12 U MNase (Worthington) to templates lacking histone H1 and 0.48 U MNase to H1-containing templates at 37°C. ..

    Activity Assay:

    Article Title: Genome-wide studies reveal novel and distinct biological pathways regulated by SIN3 isoforms
    Article Snippet: The pellet was resuspended in 200 μl MNase digest buffer (15 mM Tris-HCl pH 8, 60 mM KCl, 15 mM NaCl, 1 mM DTT, 0.25 M sucrose, 1 mM CaCl2 ) and subjected to micrococcal nuclease digestion by addition of 20 units of MNase (Worthington Biochemicals) for 10 min at room temperature. .. 10 mM EDTA was added to quench the MNase activity.

    Modification:

    Article Title: Zelda overcomes the high intrinsic nucleosome barrier at enhancers during Drosophila zygotic genome activation
    Article Snippet: Briefly, chromatin was extracted from 100–150 µL of sorted 2–3 h wt and zld − embryos per replicate, then digested with an MNase (Worthington Biochemical Corporation # ) concentration gradient of 20, 10, 5, 5/2, 5/4, 5/8, 5/16, 5/32, and 0 units (negative control) for 30 min at 37°C. .. Libraries were prepared using the NEBNext DNA Library Prep Master Mix Set for Illumina kit either following the manufacturer's instructions, or with a modified protocol , then subjected to paired-end sequencing on an Illumina HiSeq 2500 sequencing system.

    Southern Blot:

    Article Title: Coordinated repressive chromatin-remodeling of Oct4 and Nanog genes in RA-induced differentiation of embryonic stem cells involves RIP140
    Article Snippet: Nuclei isolated from ESCs were digested with 5 and 30 U of MNase (Worthington, Lakewood, NJ, USA, www.worthington-biochem.com ) at 37°C for 5 min, followed by proteinase K treatment at 37°C overnight. .. The purified DNA was subjected to Southern blot analysis.

    Article Title: Promyelocytic leukemia protein in retinoic acid-induced chromatin remodeling of Oct4 gene promoter
    Article Snippet: Nuclei isolated from P19 cells were digested with 20 and 80 U of MNase (Worthington) at 37°C for 5 min, followed by proteinase K treatment at 37°C overnight. .. The purified DNA was subjected to Southern blot analysis.

    Protease Inhibitor:

    Article Title: Switching roles for DNA and histone methylation depend on evolutionary ages of human endogenous retroviruses
    Article Snippet: Briefly, 5-aza-CdR-treated (or control) HCT116 cells (107 cells) were harvested, washed twice, and resuspended in ice-cold buffer N (15 mM Tris pH 7.5, 15 mM NaCl, 60 mM KCl, 8.5% [w/v] sucrose, 5 mM MgCl2 , 1 mM CaCl2 , 1 mM DTT, 200 μM PMSF, and 1× cOmplete Mini EDTA-free Protease Inhibitor Cocktail [Roche]). .. To isolate single nucleosomes, the nuclei were digested with MNase (1 unit Worthington MNase per 70 μg of chromatin for 10 min at 37°C).

    Article Title: Dual inhibition of DNA and histone methyltransferases increases viral mimicry in ovarian cancer cells
    Article Snippet: Briefly, A2780 cells (107 cells) before and after 5-aza-CdR, G9Ai, or combination treatment were harvested and washed twice and resuspended in ice-cold Buffer N (15 mM Tris pH7.5, 15 mM NaCl, 60 mM KCl, 8.5%(w/v) Surcose, 5 mM MgCl2 , 1 mM CaCl2 , 1 mM DTT, 200 μM PMSF, 1X cOmplete™ Mini EDTA-free Protease Inhibitor Cocktail (Roche)). .. To isolate single nucleosomes, the nuclei were digested with MNase (1U Worthington MNase per 70 μg of chromatin at 37°C for 10 min), the nucleosomes were then purified by hydroxyapatite chromatography, and adjusted to a concentration of 20 μg/mL with ChIP Buffer 1(25 mM Tris pH 7.5, 5 mM MgCl2, 100 mM KCl, 10% (v/v) glycerol, 0.1% (v/v) NP-40 substitute) and analyzed using 2% agrose gel.

    Transferring:

    Article Title: Nucleosomes containing the histone variant H2A.Bbd organize only 118 base pairs of DNA
    Article Snippet: Identical amounts of mm -146-NCP and Bbd-146-NCP (2 μg NCP per reaction) were digested with increasing amounts of MNase (Worthington) at 37°C in 125 μl of MNase buffer (0.6 mM HEPES (pH 7.6), 52 mM KCl, 5% (vol/vol) glycerol, 1.4% (wt/vol) polyethylene glycol, 5.4 mM CaCl2 ) for 1.5 min. .. The reactions were stopped by transferring to tubes containing 12.5 μl TE (10 mM Tris–HCl, 1 mM EDTA, pH 8.0), 12.5 μl 0.5 M EDTA and 200 μl stop buffer (20 mM EDTA, 200 mM NaCl, 1% (wt/vol) SDS, 0.25 mg/ml glycogen).

    Sequencing:

    Article Title: Zelda overcomes the high intrinsic nucleosome barrier at enhancers during Drosophila zygotic genome activation
    Article Snippet: Briefly, chromatin was extracted from 100–150 µL of sorted 2–3 h wt and zld − embryos per replicate, then digested with an MNase (Worthington Biochemical Corporation # ) concentration gradient of 20, 10, 5, 5/2, 5/4, 5/8, 5/16, 5/32, and 0 units (negative control) for 30 min at 37°C. .. Libraries were prepared using the NEBNext DNA Library Prep Master Mix Set for Illumina kit either following the manufacturer's instructions, or with a modified protocol , then subjected to paired-end sequencing on an Illumina HiSeq 2500 sequencing system.

    Sonication:

    Article Title: Genome-wide studies reveal novel and distinct biological pathways regulated by SIN3 isoforms
    Article Snippet: The pellet was resuspended in 200 μl MNase digest buffer (15 mM Tris-HCl pH 8, 60 mM KCl, 15 mM NaCl, 1 mM DTT, 0.25 M sucrose, 1 mM CaCl2 ) and subjected to micrococcal nuclease digestion by addition of 20 units of MNase (Worthington Biochemicals) for 10 min at room temperature. .. Following MNase digestion, samples were made up to 1.2 ml with resuspension buffer (140 mM NaCl, 10 mM Tris-HCl pH 7.6, 2 mM EDTA) and sonicated using a probe sonicator (Fisher Scientific) to extract cross-linked chromatin in the solution.

    ChIP-sequencing:

    Article Title: Switching roles for DNA and histone methylation depend on evolutionary ages of human endogenous retroviruses
    Article Snippet: Paragraph title: ChIP-seq ... To isolate single nucleosomes, the nuclei were digested with MNase (1 unit Worthington MNase per 70 μg of chromatin for 10 min at 37°C).

    In Vivo:

    Article Title: H3.3/H2A.Z double variant-containing nucleosomes mark 'nucleosome-free regions' of active promoters and other regulatory regions in the human genome
    Article Snippet: We previously showed that the instability of the variant histones seemed unaffected by acetylation , but butyrate is used during preparation to mimic the in vivo state as closely as possible. .. The A260 was adjusted to 1.25, and the resuspended nuclei were digested with 12 × 10-2 U/μL MNase (Worthington) for 10 min at 37°C to generate mostly mononucleosomes.

    Isolation:

    Article Title: Genome-wide mapping of nucleosomes in yeast
    Article Snippet: Paragraph title: Isolation of mononucleosomal DNA ... MNase: We always obtain MNase from Worthington.

    Article Title: H3.3/H2A.Z double variant-containing nucleosomes mark 'nucleosome-free regions' of active promoters and other regulatory regions in the human genome
    Article Snippet: Paragraph title: Isolation of mononucleosomes ... The A260 was adjusted to 1.25, and the resuspended nuclei were digested with 12 × 10-2 U/μL MNase (Worthington) for 10 min at 37°C to generate mostly mononucleosomes.

    Article Title: Coordinated repressive chromatin-remodeling of Oct4 and Nanog genes in RA-induced differentiation of embryonic stem cells involves RIP140
    Article Snippet: .. Nuclei isolated from ESCs were digested with 5 and 30 U of MNase (Worthington, Lakewood, NJ, USA, www.worthington-biochem.com ) at 37°C for 5 min, followed by proteinase K treatment at 37°C overnight. .. The purified DNA was subjected to Southern blot analysis.

    Article Title: Promyelocytic leukemia protein in retinoic acid-induced chromatin remodeling of Oct4 gene promoter
    Article Snippet: .. Nuclei isolated from P19 cells were digested with 20 and 80 U of MNase (Worthington) at 37°C for 5 min, followed by proteinase K treatment at 37°C overnight. .. The purified DNA was subjected to Southern blot analysis.

    Titration:

    Article Title: Hierarchical regulation of the genome: global changes in nucleosome organization potentiate genome response
    Article Snippet: .. MNase cleavage and purification of mononucleosomal and subnucleosomal DNA iSLK.219 nuclei were digested for 5 min at 37°C with a titration of MNase: 4 units/mL, 2 units/mL, and 1 unit/mL of MNase (Worthington Biochemical Corp.) in MNase cleavage buffer (4 mM MgCl2 , 5 mM KCl, 50 mM Tris-Cl (pH 7.4), 1 mM CaCl2 , 12.5% glycerol). ..

    Purification:

    Article Title: Switching roles for DNA and histone methylation depend on evolutionary ages of human endogenous retroviruses
    Article Snippet: To isolate single nucleosomes, the nuclei were digested with MNase (1 unit Worthington MNase per 70 μg of chromatin for 10 min at 37°C). .. The nucleosomes were then purified by hydroxyapatite chromatography and adjusted to a concentration of 20 μg/mL with ChIP buffer 1 (25 mM Tris pH 7.5, 5 mM MgCl2 , 100 mM KCl, 10% [v/v] glycerol, and 0.1% [v/v] NP-40 substitute) and analyzed using 2% agrose gel.

    Article Title: H3.3/H2A.Z double variant-containing nucleosomes mark 'nucleosome-free regions' of active promoters and other regulatory regions in the human genome
    Article Snippet: The A260 was adjusted to 1.25, and the resuspended nuclei were digested with 12 × 10-2 U/μL MNase (Worthington) for 10 min at 37°C to generate mostly mononucleosomes. .. Mononucleosomes were then purified on a 5%–30% sucrose gradient containing 10 mM NaCl, 10 mM Tris-HCl (pH 7.4), and 0.2 mM EDTA.

    Article Title: Dual inhibition of DNA and histone methyltransferases increases viral mimicry in ovarian cancer cells
    Article Snippet: .. To isolate single nucleosomes, the nuclei were digested with MNase (1U Worthington MNase per 70 μg of chromatin at 37°C for 10 min), the nucleosomes were then purified by hydroxyapatite chromatography, and adjusted to a concentration of 20 μg/mL with ChIP Buffer 1(25 mM Tris pH 7.5, 5 mM MgCl2, 100 mM KCl, 10% (v/v) glycerol, 0.1% (v/v) NP-40 substitute) and analyzed using 2% agrose gel. .. H3K9me2, H3K4me3 and H3K27ac ChIP were performed as previously described , using 5 μg of nucleosome pulled down with 10 μg of anti-H3K9me2 (Abcam ab1220), anti-H3K4me3 (Abcam ab1012), and anti-H3K27ac (Active Motif AM39133) antibody on Dynabeads Protein G (Invitrogen) for 2 h at 4°C.

    Article Title: Coordinated repressive chromatin-remodeling of Oct4 and Nanog genes in RA-induced differentiation of embryonic stem cells involves RIP140
    Article Snippet: Nuclei isolated from ESCs were digested with 5 and 30 U of MNase (Worthington, Lakewood, NJ, USA, www.worthington-biochem.com ) at 37°C for 5 min, followed by proteinase K treatment at 37°C overnight. .. The purified DNA was subjected to Southern blot analysis.

    Article Title: Hierarchical regulation of the genome: global changes in nucleosome organization potentiate genome response
    Article Snippet: .. MNase cleavage and purification of mononucleosomal and subnucleosomal DNA iSLK.219 nuclei were digested for 5 min at 37°C with a titration of MNase: 4 units/mL, 2 units/mL, and 1 unit/mL of MNase (Worthington Biochemical Corp.) in MNase cleavage buffer (4 mM MgCl2 , 5 mM KCl, 50 mM Tris-Cl (pH 7.4), 1 mM CaCl2 , 12.5% glycerol). ..

    Article Title: Promyelocytic leukemia protein in retinoic acid-induced chromatin remodeling of Oct4 gene promoter
    Article Snippet: Nuclei isolated from P19 cells were digested with 20 and 80 U of MNase (Worthington) at 37°C for 5 min, followed by proteinase K treatment at 37°C overnight. .. The purified DNA was subjected to Southern blot analysis.

    Article Title: Lysine methyltransferase G9a is not required for DNMT3A/3B anchoring to methylated nucleosomes and maintenance of DNA methylation in somatic cells
    Article Snippet: .. Purified nuclei (1x108 ) resuspended in 1 ml of RSB containing 0.25 M sucrose, 3 mM CaCl2 , and 100 μM PMSF, were digested with 36 units of MNase (Worthington) for partial and 500 units of MNase (Worthington) for extensive digestion for 15 min at 37°C, and then the reaction was stopped with EDTA/EGTA (up to 10 mM). ..

    Chromatin Immunoprecipitation:

    Article Title: Switching roles for DNA and histone methylation depend on evolutionary ages of human endogenous retroviruses
    Article Snippet: Single nucleosome preparation was performed according to the Dilworth laboratory native ChIP protocol ( ). .. To isolate single nucleosomes, the nuclei were digested with MNase (1 unit Worthington MNase per 70 μg of chromatin for 10 min at 37°C).

    Article Title: Dual inhibition of DNA and histone methyltransferases increases viral mimicry in ovarian cancer cells
    Article Snippet: .. To isolate single nucleosomes, the nuclei were digested with MNase (1U Worthington MNase per 70 μg of chromatin at 37°C for 10 min), the nucleosomes were then purified by hydroxyapatite chromatography, and adjusted to a concentration of 20 μg/mL with ChIP Buffer 1(25 mM Tris pH 7.5, 5 mM MgCl2, 100 mM KCl, 10% (v/v) glycerol, 0.1% (v/v) NP-40 substitute) and analyzed using 2% agrose gel. .. H3K9me2, H3K4me3 and H3K27ac ChIP were performed as previously described , using 5 μg of nucleosome pulled down with 10 μg of anti-H3K9me2 (Abcam ab1220), anti-H3K4me3 (Abcam ab1012), and anti-H3K27ac (Active Motif AM39133) antibody on Dynabeads Protein G (Invitrogen) for 2 h at 4°C.

    Negative Control:

    Article Title: Zelda overcomes the high intrinsic nucleosome barrier at enhancers during Drosophila zygotic genome activation
    Article Snippet: .. Briefly, chromatin was extracted from 100–150 µL of sorted 2–3 h wt and zld − embryos per replicate, then digested with an MNase (Worthington Biochemical Corporation # ) concentration gradient of 20, 10, 5, 5/2, 5/4, 5/8, 5/16, 5/32, and 0 units (negative control) for 30 min at 37°C. .. Mononucleosome-sized DNA was extracted from lanes containing 20 and 10 units MNase digested samples on a 1.7% agarose gel, when the dinucleosome-sized DNA band just disappeared, indicating saturation but not overdigestion.

    Gradient Centrifugation:

    Article Title: Lysine methyltransferase G9a is not required for DNMT3A/3B anchoring to methylated nucleosomes and maintenance of DNA methylation in somatic cells
    Article Snippet: Paragraph title: MNase digestion and sucrose density gradient centrifugation ... Purified nuclei (1x108 ) resuspended in 1 ml of RSB containing 0.25 M sucrose, 3 mM CaCl2 , and 100 μM PMSF, were digested with 36 units of MNase (Worthington) for partial and 500 units of MNase (Worthington) for extensive digestion for 15 min at 37°C, and then the reaction was stopped with EDTA/EGTA (up to 10 mM).

    Agarose Gel Electrophoresis:

    Article Title: Zelda overcomes the high intrinsic nucleosome barrier at enhancers during Drosophila zygotic genome activation
    Article Snippet: Briefly, chromatin was extracted from 100–150 µL of sorted 2–3 h wt and zld − embryos per replicate, then digested with an MNase (Worthington Biochemical Corporation # ) concentration gradient of 20, 10, 5, 5/2, 5/4, 5/8, 5/16, 5/32, and 0 units (negative control) for 30 min at 37°C. .. Mononucleosome-sized DNA was extracted from lanes containing 20 and 10 units MNase digested samples on a 1.7% agarose gel, when the dinucleosome-sized DNA band just disappeared, indicating saturation but not overdigestion.

    Article Title: Hierarchical regulation of the genome: global changes in nucleosome organization potentiate genome response
    Article Snippet: MNase cleavage and purification of mononucleosomal and subnucleosomal DNA iSLK.219 nuclei were digested for 5 min at 37°C with a titration of MNase: 4 units/mL, 2 units/mL, and 1 unit/mL of MNase (Worthington Biochemical Corp.) in MNase cleavage buffer (4 mM MgCl2 , 5 mM KCl, 50 mM Tris-Cl (pH 7.4), 1 mM CaCl2 , 12.5% glycerol). .. The samples were then run and the nucleosomal ladder was separated on a 2% agarose gel.

    Chromatography:

    Article Title: Switching roles for DNA and histone methylation depend on evolutionary ages of human endogenous retroviruses
    Article Snippet: To isolate single nucleosomes, the nuclei were digested with MNase (1 unit Worthington MNase per 70 μg of chromatin for 10 min at 37°C). .. The nucleosomes were then purified by hydroxyapatite chromatography and adjusted to a concentration of 20 μg/mL with ChIP buffer 1 (25 mM Tris pH 7.5, 5 mM MgCl2 , 100 mM KCl, 10% [v/v] glycerol, and 0.1% [v/v] NP-40 substitute) and analyzed using 2% agrose gel.

    Article Title: Dual inhibition of DNA and histone methyltransferases increases viral mimicry in ovarian cancer cells
    Article Snippet: .. To isolate single nucleosomes, the nuclei were digested with MNase (1U Worthington MNase per 70 μg of chromatin at 37°C for 10 min), the nucleosomes were then purified by hydroxyapatite chromatography, and adjusted to a concentration of 20 μg/mL with ChIP Buffer 1(25 mM Tris pH 7.5, 5 mM MgCl2, 100 mM KCl, 10% (v/v) glycerol, 0.1% (v/v) NP-40 substitute) and analyzed using 2% agrose gel. .. H3K9me2, H3K4me3 and H3K27ac ChIP were performed as previously described , using 5 μg of nucleosome pulled down with 10 μg of anti-H3K9me2 (Abcam ab1220), anti-H3K4me3 (Abcam ab1012), and anti-H3K27ac (Active Motif AM39133) antibody on Dynabeads Protein G (Invitrogen) for 2 h at 4°C.

    Ethanol Precipitation:

    Article Title: Tax Abolishes Histone H1 Repression of p300 Acetyltransferase Activity at the Human T-Cell Leukemia Virus Type 1 Promoter ▿
    Article Snippet: First, 135 μl of MNase buffer (1.74 mM HEPES [pH 7.6], 121 mM KCl, 12% [vol/vol] glycerol, 2.4% [wt/vol] polyethylene glycol, 11.2 mM CaCl2 ) were added to chromatin samples and incubated at 37°C for 5 min. Digestion was initiated by addition of 0.12 U MNase (Worthington) to templates lacking histone H1 and 0.48 U MNase to H1-containing templates at 37°C. .. The DNA was extracted with phenol-chloroform followed by ethanol precipitation.

    Concentration Assay:

    Article Title: Subtelomeric proteins negatively regulate telomere elongation in budding yeast
    Article Snippet: .. MNase (Worthington) or DNase I (Sigma) was added at 50 or 12.5 U/ml final concentration, respectively. ..

    Article Title: Switching roles for DNA and histone methylation depend on evolutionary ages of human endogenous retroviruses
    Article Snippet: To isolate single nucleosomes, the nuclei were digested with MNase (1 unit Worthington MNase per 70 μg of chromatin for 10 min at 37°C). .. The nucleosomes were then purified by hydroxyapatite chromatography and adjusted to a concentration of 20 μg/mL with ChIP buffer 1 (25 mM Tris pH 7.5, 5 mM MgCl2 , 100 mM KCl, 10% [v/v] glycerol, and 0.1% [v/v] NP-40 substitute) and analyzed using 2% agrose gel.

    Article Title: H3.3/H2A.Z double variant-containing nucleosomes mark 'nucleosome-free regions' of active promoters and other regulatory regions in the human genome
    Article Snippet: The A260 was adjusted to 1.25, and the resuspended nuclei were digested with 12 × 10-2 U/μL MNase (Worthington) for 10 min at 37°C to generate mostly mononucleosomes. .. The reaction was stopped by adding EDTA (pH 8.0) to a final concentration of 10 mM, and the suspension was centrifuged at 2500 rpm for 5 min, retaining supernatant S1.

    Article Title: Dual inhibition of DNA and histone methyltransferases increases viral mimicry in ovarian cancer cells
    Article Snippet: .. To isolate single nucleosomes, the nuclei were digested with MNase (1U Worthington MNase per 70 μg of chromatin at 37°C for 10 min), the nucleosomes were then purified by hydroxyapatite chromatography, and adjusted to a concentration of 20 μg/mL with ChIP Buffer 1(25 mM Tris pH 7.5, 5 mM MgCl2, 100 mM KCl, 10% (v/v) glycerol, 0.1% (v/v) NP-40 substitute) and analyzed using 2% agrose gel. .. H3K9me2, H3K4me3 and H3K27ac ChIP were performed as previously described , using 5 μg of nucleosome pulled down with 10 μg of anti-H3K9me2 (Abcam ab1220), anti-H3K4me3 (Abcam ab1012), and anti-H3K27ac (Active Motif AM39133) antibody on Dynabeads Protein G (Invitrogen) for 2 h at 4°C.

    Article Title: Zelda overcomes the high intrinsic nucleosome barrier at enhancers during Drosophila zygotic genome activation
    Article Snippet: .. Briefly, chromatin was extracted from 100–150 µL of sorted 2–3 h wt and zld − embryos per replicate, then digested with an MNase (Worthington Biochemical Corporation # ) concentration gradient of 20, 10, 5, 5/2, 5/4, 5/8, 5/16, 5/32, and 0 units (negative control) for 30 min at 37°C. .. Mononucleosome-sized DNA was extracted from lanes containing 20 and 10 units MNase digested samples on a 1.7% agarose gel, when the dinucleosome-sized DNA band just disappeared, indicating saturation but not overdigestion.

    Article Title: Genome-wide studies reveal novel and distinct biological pathways regulated by SIN3 isoforms
    Article Snippet: Excess formaldehyde was quenched by addition of glycine to a final concentration of 125 mM. .. The pellet was resuspended in 200 μl MNase digest buffer (15 mM Tris-HCl pH 8, 60 mM KCl, 15 mM NaCl, 1 mM DTT, 0.25 M sucrose, 1 mM CaCl2 ) and subjected to micrococcal nuclease digestion by addition of 20 units of MNase (Worthington Biochemicals) for 10 min at room temperature.

    Lysis:

    Article Title: Switching roles for DNA and histone methylation depend on evolutionary ages of human endogenous retroviruses
    Article Snippet: To prepare nuclei, cells were lysed in 1 mL lysis buffer (buffer N supplemented with 0.3% NP-40 substitute [Sigma]) for 10 min at 4°C, and nuclei were collected by centrifugation (500 g for 5 min at 4°C), resuspended in 1 mL of buffer N, and then sedimented through 7.5 mL sucrose cushion (10 mM HEPES pH 7.9, 30% [w/v] sucrose, 1.5 mM MgCl2 ) at 13,000 g using a Sorvall swinging bucket for 12 min at 4°C. .. To isolate single nucleosomes, the nuclei were digested with MNase (1 unit Worthington MNase per 70 μg of chromatin for 10 min at 37°C).

    Article Title: H3.3/H2A.Z double variant-containing nucleosomes mark 'nucleosome-free regions' of active promoters and other regulatory regions in the human genome
    Article Snippet: Isolation of mononucleosomes Nuclei were isolated with a cell lysis buffer containing 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2 , and 0.4% NP-40. .. The A260 was adjusted to 1.25, and the resuspended nuclei were digested with 12 × 10-2 U/μL MNase (Worthington) for 10 min at 37°C to generate mostly mononucleosomes.

    Article Title: Dual inhibition of DNA and histone methyltransferases increases viral mimicry in ovarian cancer cells
    Article Snippet: To prepare nuclei, cells were lysed in 1 mL Lysis Buffer (Buffer N supplemented with 0.3% NP-40 substitute (Sigma)) for 10 min at 4°C, and nuclei were collected by centrifugation (500 × g for 5 min at 4°C), resuspended in 1 mL of Buffer N, then sedimented through 7.5 mL sucrose cushion (10 mM HEPES pH7.9, 30%(w/v) sucrose, 1.5 mM MgCl2 and centrifuged 13000 × g using Sorvall swinging bucket for 12 min at 4°C). .. To isolate single nucleosomes, the nuclei were digested with MNase (1U Worthington MNase per 70 μg of chromatin at 37°C for 10 min), the nucleosomes were then purified by hydroxyapatite chromatography, and adjusted to a concentration of 20 μg/mL with ChIP Buffer 1(25 mM Tris pH 7.5, 5 mM MgCl2, 100 mM KCl, 10% (v/v) glycerol, 0.1% (v/v) NP-40 substitute) and analyzed using 2% agrose gel.

    Article Title: Genome-wide studies reveal novel and distinct biological pathways regulated by SIN3 isoforms
    Article Snippet: The cross-linked cells were washed with 1X phosphate buffered saline and resuspended in lysis buffer (10 mM Tris-HCl pH 8, 10 mM KCl, 3 mM CaCl2 , 0.34 M sucrose, 1 mM DTT, 0.1 % Triton X-100, 0.2 mM EGTA, proteinase inhibitor (Roche)), followed by incubation on ice for 15 min. .. The pellet was resuspended in 200 μl MNase digest buffer (15 mM Tris-HCl pH 8, 60 mM KCl, 15 mM NaCl, 1 mM DTT, 0.25 M sucrose, 1 mM CaCl2 ) and subjected to micrococcal nuclease digestion by addition of 20 units of MNase (Worthington Biochemicals) for 10 min at room temperature.

    Variant Assay:

    Article Title: H3.3/H2A.Z double variant-containing nucleosomes mark 'nucleosome-free regions' of active promoters and other regulatory regions in the human genome
    Article Snippet: We previously showed that the instability of the variant histones seemed unaffected by acetylation , but butyrate is used during preparation to mimic the in vivo state as closely as possible. .. The A260 was adjusted to 1.25, and the resuspended nuclei were digested with 12 × 10-2 U/μL MNase (Worthington) for 10 min at 37°C to generate mostly mononucleosomes.

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    Worthington Biochemical mnase
    RSC Maintains Open NFRs in Lowly Expressed Genes but Is Not Necessary for an Acute Transcriptional Response (A) Experiment outline (see Figure 2 A). (B) RNA fold change during Sth1 depletion and recovery. RNA level was normalized with K. lactis spike-in. Each row is a gene (5,529 genes), and each column is a sample. Heatmap is normalized to expression level prior to auxin addition (also mid-log). The levels of genes at this time are shown by the orange and purple columns. (C) NFR width per RNA level. NFR width per RNA percentile in each sample (Loess smoothed) (top). Percentage of NFRs that closed in the presence of auxin for 0.5 hr (orange line) and 2 hr (yellow line) out of the NFRs that were open in steady state, per RNA percentile at the same time point (bottom). (D) Stress experiment outline. Yeast cells were grown to mid-log in YPD. Auxin was added for 20 min, followed by salt addition (0.4 M KCl); samples were taken in time course and were subjected to <t>MNase-seq</t> and RNA sequencing (RNA-seq). Control samples without auxin or without KCL were performed. (E) Heatmap of RNA fold change in three treatments: auxin only, salt only, and both salt and auxin. RNA levels are normalized per library. 2,322 clustered genes that change in response to the treatments are shown as fold change with respect to the matching expression at T = 0. Time points are indicated in the experiment outline (A). (F) Metagene of subsets of stress-induced genes showing a typical response of chromatin structure to salt induction in time points in three treatments: auxin only, KCl only, and both KCl and auxin. Genes are positioned according to the nucleosome +1 center at T = 0. Black arrows mark location of changes. See also Figure S4 .
    Mnase, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 96/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RSC Maintains Open NFRs in Lowly Expressed Genes but Is Not Necessary for an Acute Transcriptional Response (A) Experiment outline (see Figure 2 A). (B) RNA fold change during Sth1 depletion and recovery. RNA level was normalized with K. lactis spike-in. Each row is a gene (5,529 genes), and each column is a sample. Heatmap is normalized to expression level prior to auxin addition (also mid-log). The levels of genes at this time are shown by the orange and purple columns. (C) NFR width per RNA level. NFR width per RNA percentile in each sample (Loess smoothed) (top). Percentage of NFRs that closed in the presence of auxin for 0.5 hr (orange line) and 2 hr (yellow line) out of the NFRs that were open in steady state, per RNA percentile at the same time point (bottom). (D) Stress experiment outline. Yeast cells were grown to mid-log in YPD. Auxin was added for 20 min, followed by salt addition (0.4 M KCl); samples were taken in time course and were subjected to MNase-seq and RNA sequencing (RNA-seq). Control samples without auxin or without KCL were performed. (E) Heatmap of RNA fold change in three treatments: auxin only, salt only, and both salt and auxin. RNA levels are normalized per library. 2,322 clustered genes that change in response to the treatments are shown as fold change with respect to the matching expression at T = 0. Time points are indicated in the experiment outline (A). (F) Metagene of subsets of stress-induced genes showing a typical response of chromatin structure to salt induction in time points in three treatments: auxin only, KCl only, and both KCl and auxin. Genes are positioned according to the nucleosome +1 center at T = 0. Black arrows mark location of changes. See also Figure S4 .

    Journal: Cell Reports

    Article Title: Dynamics of Chromatin and Transcription during Transient Depletion of the RSC Chromatin Remodeling Complex

    doi: 10.1016/j.celrep.2018.12.020

    Figure Lengend Snippet: RSC Maintains Open NFRs in Lowly Expressed Genes but Is Not Necessary for an Acute Transcriptional Response (A) Experiment outline (see Figure 2 A). (B) RNA fold change during Sth1 depletion and recovery. RNA level was normalized with K. lactis spike-in. Each row is a gene (5,529 genes), and each column is a sample. Heatmap is normalized to expression level prior to auxin addition (also mid-log). The levels of genes at this time are shown by the orange and purple columns. (C) NFR width per RNA level. NFR width per RNA percentile in each sample (Loess smoothed) (top). Percentage of NFRs that closed in the presence of auxin for 0.5 hr (orange line) and 2 hr (yellow line) out of the NFRs that were open in steady state, per RNA percentile at the same time point (bottom). (D) Stress experiment outline. Yeast cells were grown to mid-log in YPD. Auxin was added for 20 min, followed by salt addition (0.4 M KCl); samples were taken in time course and were subjected to MNase-seq and RNA sequencing (RNA-seq). Control samples without auxin or without KCL were performed. (E) Heatmap of RNA fold change in three treatments: auxin only, salt only, and both salt and auxin. RNA levels are normalized per library. 2,322 clustered genes that change in response to the treatments are shown as fold change with respect to the matching expression at T = 0. Time points are indicated in the experiment outline (A). (F) Metagene of subsets of stress-induced genes showing a typical response of chromatin structure to salt induction in time points in three treatments: auxin only, KCl only, and both KCl and auxin. Genes are positioned according to the nucleosome +1 center at T = 0. Black arrows mark location of changes. See also Figure S4 .

    Article Snippet: Spheroplasts were washed, resuspended in NP buffer and treated with MNase (Micrococcal nuclease, Worthington) to generate 80% mono-nucleosomes (1 unit for 2.5 OD initial culture, 37°C, 20 minutes).

    Techniques: Expressing, RNA Sequencing Assay

    Changes in the +1 Nucleosome Position Are Reflected in TSS Usage (A) Experimental outline (as in Figure 2 A). An example of the data representation showing RNA 5′ ends (black), MNase read centers (dark red), and coverage (light red) around the TSS. (B) Nucleosome positioning and 5′ RNA ends during Sth1 depletion in CDC8 and ATG27 promoters. Dashed lines represent peak centers before and 1 hr after auxin addition. (C) 5′ RNA level at each position over the genome before and after Sth1 depletion (normalized with K. lactis spike-in). (D) Median nucleosome positioning around mRNA 5′ ends before (top) and 1 hr after (bottom) auxin addition. mRNA 5′ positions are separated to groups according to their fold change following Sth1 depletion. (E) Change in expression (1 hr/0 hr) versus change in accessibility (1 hr/0 hr) for mRNA 5′ locations that are expressed ( Figure 5 C) and accessible ( Figure S5 B) before auxin addition. See also Figure S5 .

    Journal: Cell Reports

    Article Title: Dynamics of Chromatin and Transcription during Transient Depletion of the RSC Chromatin Remodeling Complex

    doi: 10.1016/j.celrep.2018.12.020

    Figure Lengend Snippet: Changes in the +1 Nucleosome Position Are Reflected in TSS Usage (A) Experimental outline (as in Figure 2 A). An example of the data representation showing RNA 5′ ends (black), MNase read centers (dark red), and coverage (light red) around the TSS. (B) Nucleosome positioning and 5′ RNA ends during Sth1 depletion in CDC8 and ATG27 promoters. Dashed lines represent peak centers before and 1 hr after auxin addition. (C) 5′ RNA level at each position over the genome before and after Sth1 depletion (normalized with K. lactis spike-in). (D) Median nucleosome positioning around mRNA 5′ ends before (top) and 1 hr after (bottom) auxin addition. mRNA 5′ positions are separated to groups according to their fold change following Sth1 depletion. (E) Change in expression (1 hr/0 hr) versus change in accessibility (1 hr/0 hr) for mRNA 5′ locations that are expressed ( Figure 5 C) and accessible ( Figure S5 B) before auxin addition. See also Figure S5 .

    Article Snippet: Spheroplasts were washed, resuspended in NP buffer and treated with MNase (Micrococcal nuclease, Worthington) to generate 80% mono-nucleosomes (1 unit for 2.5 OD initial culture, 37°C, 20 minutes).

    Techniques: Expressing

    Dynamics of Sth1 Depletion and Recovery Show Massive yet Reversible Disruptions in Chromatin Organization (A) Experimental outline. For depletion, auxin (IAA) was added to mid-log degron-Sth1 cells, and MNase-seq was performed at the indicated time points. For recovery, mid-log degron-Sth1 cells were incubated in the presence of auxin for 2 hr. Auxin was washed from the media, and MNase-seq was performed at the indicated time points. (B) Median MNase coverage positioned relative to the TSS (metagene) following Sth1 depletion (top) and recovery (bottom). (C) MNase read centers (lines, dark color) and coverage (shade, light color) following Sth1 depletion and recovery in the TAF6/NSA1 promoter area. Dashed lines represent the position of nucleosomes +1 and −1 center before depletion and after full recovery. (D) Distribution of NFR width (defined as the distance between the peak −/+1 nucleosomes) through sth1 depletion and recovery. (E) Average MNase coverage (metagene) before (red line) and 1 hr after (yellow line) Sth1 depletion in genes with a GRF-binding site (top) and without GRF binding but with a poly(A/T) tract (bottom). Genes were positioned relative to the GRF-binding site or poly(A/T) tract site. GRF-binding sites were obtained from Gutin et al. (2018) . (F) Distribution of NFR width throughout Sth1 depletion in the two groups as in (E). The distribution of all genes before auxin addition is shown in gray. (G) Comparison of NFR width before Sth1 depletion and after recovery. Each point is related to NFR of a gene. Genes with fuzzy +1 or −1 nucleosomes were excluded. See also Figure S2 .

    Journal: Cell Reports

    Article Title: Dynamics of Chromatin and Transcription during Transient Depletion of the RSC Chromatin Remodeling Complex

    doi: 10.1016/j.celrep.2018.12.020

    Figure Lengend Snippet: Dynamics of Sth1 Depletion and Recovery Show Massive yet Reversible Disruptions in Chromatin Organization (A) Experimental outline. For depletion, auxin (IAA) was added to mid-log degron-Sth1 cells, and MNase-seq was performed at the indicated time points. For recovery, mid-log degron-Sth1 cells were incubated in the presence of auxin for 2 hr. Auxin was washed from the media, and MNase-seq was performed at the indicated time points. (B) Median MNase coverage positioned relative to the TSS (metagene) following Sth1 depletion (top) and recovery (bottom). (C) MNase read centers (lines, dark color) and coverage (shade, light color) following Sth1 depletion and recovery in the TAF6/NSA1 promoter area. Dashed lines represent the position of nucleosomes +1 and −1 center before depletion and after full recovery. (D) Distribution of NFR width (defined as the distance between the peak −/+1 nucleosomes) through sth1 depletion and recovery. (E) Average MNase coverage (metagene) before (red line) and 1 hr after (yellow line) Sth1 depletion in genes with a GRF-binding site (top) and without GRF binding but with a poly(A/T) tract (bottom). Genes were positioned relative to the GRF-binding site or poly(A/T) tract site. GRF-binding sites were obtained from Gutin et al. (2018) . (F) Distribution of NFR width throughout Sth1 depletion in the two groups as in (E). The distribution of all genes before auxin addition is shown in gray. (G) Comparison of NFR width before Sth1 depletion and after recovery. Each point is related to NFR of a gene. Genes with fuzzy +1 or −1 nucleosomes were excluded. See also Figure S2 .

    Article Snippet: Spheroplasts were washed, resuspended in NP buffer and treated with MNase (Micrococcal nuclease, Worthington) to generate 80% mono-nucleosomes (1 unit for 2.5 OD initial culture, 37°C, 20 minutes).

    Techniques: Incubation, Binding Assay

    Sth1-Dependent NFR Clearing Is Replication Independent (A) Experimental outline in G1-arrested cells. For depletion, yeast cells were grown to mid-log in YPD and incubated with or without alpha factor for 2 hr. At the indicated time, cells were transferred to a new tube, and Sth1 depletion was induced by auxin addition. All samples were fixed at the same time. For recovery, yeast cells were grown to mid-log in YPD and incubated with alpha-factor and auxin for 2 hr. Cells were washed and resuspended with or without alpha factor. MNase-seq was performed at the indicated time points. (B) Distribution of NFR width in time course through Sth1 depletion (top) and recovery (bottom) in G1-arrested cells (right) and in unsynchronized cells (left). (C) Density scatter of the change in NFR width for all genes through Sth1 depletion (1 hr, top) and recovery (4 hr, bottom), in G1 arrested versus unsynchronized cells. See also Figure S3 .

    Journal: Cell Reports

    Article Title: Dynamics of Chromatin and Transcription during Transient Depletion of the RSC Chromatin Remodeling Complex

    doi: 10.1016/j.celrep.2018.12.020

    Figure Lengend Snippet: Sth1-Dependent NFR Clearing Is Replication Independent (A) Experimental outline in G1-arrested cells. For depletion, yeast cells were grown to mid-log in YPD and incubated with or without alpha factor for 2 hr. At the indicated time, cells were transferred to a new tube, and Sth1 depletion was induced by auxin addition. All samples were fixed at the same time. For recovery, yeast cells were grown to mid-log in YPD and incubated with alpha-factor and auxin for 2 hr. Cells were washed and resuspended with or without alpha factor. MNase-seq was performed at the indicated time points. (B) Distribution of NFR width in time course through Sth1 depletion (top) and recovery (bottom) in G1-arrested cells (right) and in unsynchronized cells (left). (C) Density scatter of the change in NFR width for all genes through Sth1 depletion (1 hr, top) and recovery (4 hr, bottom), in G1 arrested versus unsynchronized cells. See also Figure S3 .

    Article Snippet: Spheroplasts were washed, resuspended in NP buffer and treated with MNase (Micrococcal nuclease, Worthington) to generate 80% mono-nucleosomes (1 unit for 2.5 OD initial culture, 37°C, 20 minutes).

    Techniques: Incubation

    Induced Knockdown Screen of ATP-Dependent Chromatin Remodelers (A) An auxin-inducible degron (AID) system ( Morawska and Ulrich, 2013 ) yielding an auxin-inducible, rapid degradation of tagged chromatin remodelers. Plant hormone auxin (IAA) directly induces rapid degradation of the AID-tagged protein by mediating the interaction of a degron domain in the target protein with the substrate recognition domain of TIR1. (B) Experimental outline. AID-tagged chromatin remodeler strains were grown to mid-log in YPD. MNase-seq was performed to compare nucleosome positioning before and at two time points after auxin addition. (C) Average MNase coverage positioned relative to the transcription start site (TSS) (“metagene”) for each chromatin remodeler AID strain before and after auxin addition and in the relevant KO strains (if available) (top). Average of the change in MNase coverage before and after auxin addition (1 hr to 0 hr) positioned relative to the TSS for each chromatin remodeler AID strain (bottom). (D) Heatmaps representing the change in MNase coverage before and after auxin addition (1 hr to 0 hr) positioned relative to the TSS (in yellow) for each AID strain. Genes (rows) are sorted, in each strain, by the magnitude of changes in coverage following the depletion in the NFR area. See also Figure S1 .

    Journal: Cell Reports

    Article Title: Dynamics of Chromatin and Transcription during Transient Depletion of the RSC Chromatin Remodeling Complex

    doi: 10.1016/j.celrep.2018.12.020

    Figure Lengend Snippet: Induced Knockdown Screen of ATP-Dependent Chromatin Remodelers (A) An auxin-inducible degron (AID) system ( Morawska and Ulrich, 2013 ) yielding an auxin-inducible, rapid degradation of tagged chromatin remodelers. Plant hormone auxin (IAA) directly induces rapid degradation of the AID-tagged protein by mediating the interaction of a degron domain in the target protein with the substrate recognition domain of TIR1. (B) Experimental outline. AID-tagged chromatin remodeler strains were grown to mid-log in YPD. MNase-seq was performed to compare nucleosome positioning before and at two time points after auxin addition. (C) Average MNase coverage positioned relative to the transcription start site (TSS) (“metagene”) for each chromatin remodeler AID strain before and after auxin addition and in the relevant KO strains (if available) (top). Average of the change in MNase coverage before and after auxin addition (1 hr to 0 hr) positioned relative to the TSS for each chromatin remodeler AID strain (bottom). (D) Heatmaps representing the change in MNase coverage before and after auxin addition (1 hr to 0 hr) positioned relative to the TSS (in yellow) for each AID strain. Genes (rows) are sorted, in each strain, by the magnitude of changes in coverage following the depletion in the NFR area. See also Figure S1 .

    Article Snippet: Spheroplasts were washed, resuspended in NP buffer and treated with MNase (Micrococcal nuclease, Worthington) to generate 80% mono-nucleosomes (1 unit for 2.5 OD initial culture, 37°C, 20 minutes).

    Techniques:

    Dynamics of Sth1 Depletion and Recovery Show Massive yet Reversible Disruptions in Chromatin Organization (A) Experimental outline. For depletion, auxin (IAA) was added to mid-log degron-Sth1 cells, and MNase-seq was performed at the indicated time points. For recovery, mid-log degron-Sth1 cells were incubated in the presence of auxin for 2 hr. Auxin was washed from the media, and MNase-seq was performed at the indicated time points. (B) Median MNase coverage positioned relative to the TSS (metagene) following Sth1 depletion (top) and recovery (bottom). (C) MNase read centers (lines, dark color) and coverage (shade, light color) following Sth1 depletion and recovery in the TAF6/NSA1 promoter area. Dashed lines represent the position of nucleosomes +1 and −1 center before depletion and after full recovery. (D) Distribution of NFR width (defined as the distance between the peak −/+1 nucleosomes) through sth1 depletion and recovery. . (F) Distribution of NFR width throughout Sth1 depletion in the two groups as in (E). The distribution of all genes before auxin addition is shown in gray. (G) Comparison of NFR width before Sth1 depletion and after recovery. Each point is related to NFR of a gene. Genes with fuzzy +1 or −1 nucleosomes were excluded. .

    Journal: Cell Reports

    Article Title: Dynamics of Chromatin and Transcription during Transient Depletion of the RSC Chromatin Remodeling Complex

    doi: 10.1016/j.celrep.2018.12.020

    Figure Lengend Snippet: Dynamics of Sth1 Depletion and Recovery Show Massive yet Reversible Disruptions in Chromatin Organization (A) Experimental outline. For depletion, auxin (IAA) was added to mid-log degron-Sth1 cells, and MNase-seq was performed at the indicated time points. For recovery, mid-log degron-Sth1 cells were incubated in the presence of auxin for 2 hr. Auxin was washed from the media, and MNase-seq was performed at the indicated time points. (B) Median MNase coverage positioned relative to the TSS (metagene) following Sth1 depletion (top) and recovery (bottom). (C) MNase read centers (lines, dark color) and coverage (shade, light color) following Sth1 depletion and recovery in the TAF6/NSA1 promoter area. Dashed lines represent the position of nucleosomes +1 and −1 center before depletion and after full recovery. (D) Distribution of NFR width (defined as the distance between the peak −/+1 nucleosomes) through sth1 depletion and recovery. . (F) Distribution of NFR width throughout Sth1 depletion in the two groups as in (E). The distribution of all genes before auxin addition is shown in gray. (G) Comparison of NFR width before Sth1 depletion and after recovery. Each point is related to NFR of a gene. Genes with fuzzy +1 or −1 nucleosomes were excluded. .

    Article Snippet: Spheroplasts were washed, resuspended in NP buffer and treated with MNase (Micrococcal nuclease, Worthington) to generate 80% mono-nucleosomes (1 unit for 2.5 OD initial culture, 37°C, 20 minutes).

    Techniques: Incubation

    Sth1-Dependent NFR Clearing Is Replication Independent (A) Experimental outline in G1-arrested cells. For depletion, yeast cells were grown to mid-log in YPD and incubated with or without alpha factor for 2 hr. At the indicated time, cells were transferred to a new tube, and Sth1 depletion was induced by auxin addition. All samples were fixed at the same time. For recovery, yeast cells were grown to mid-log in YPD and incubated with alpha-factor and auxin for 2 hr. Cells were washed and resuspended with or without alpha factor. MNase-seq was performed at the indicated time points. (B) Distribution of NFR width in time course through Sth1 depletion (top) and recovery (bottom) in G1-arrested cells (right) and in unsynchronized cells (left). (C) Density scatter of the change in NFR width for all genes through Sth1 depletion (1 hr, top) and recovery (4 hr, bottom), in G1 arrested versus unsynchronized cells. .

    Journal: Cell Reports

    Article Title: Dynamics of Chromatin and Transcription during Transient Depletion of the RSC Chromatin Remodeling Complex

    doi: 10.1016/j.celrep.2018.12.020

    Figure Lengend Snippet: Sth1-Dependent NFR Clearing Is Replication Independent (A) Experimental outline in G1-arrested cells. For depletion, yeast cells were grown to mid-log in YPD and incubated with or without alpha factor for 2 hr. At the indicated time, cells were transferred to a new tube, and Sth1 depletion was induced by auxin addition. All samples were fixed at the same time. For recovery, yeast cells were grown to mid-log in YPD and incubated with alpha-factor and auxin for 2 hr. Cells were washed and resuspended with or without alpha factor. MNase-seq was performed at the indicated time points. (B) Distribution of NFR width in time course through Sth1 depletion (top) and recovery (bottom) in G1-arrested cells (right) and in unsynchronized cells (left). (C) Density scatter of the change in NFR width for all genes through Sth1 depletion (1 hr, top) and recovery (4 hr, bottom), in G1 arrested versus unsynchronized cells. .

    Article Snippet: Spheroplasts were washed, resuspended in NP buffer and treated with MNase (Micrococcal nuclease, Worthington) to generate 80% mono-nucleosomes (1 unit for 2.5 OD initial culture, 37°C, 20 minutes).

    Techniques: Incubation

    Induced Knockdown Screen of ATP-Dependent Chromatin Remodelers ) yielding an auxin-inducible, rapid degradation of tagged chromatin remodelers. Plant hormone auxin (IAA) directly induces rapid degradation of the AID-tagged protein by mediating the interaction of a degron domain in the target protein with the substrate recognition domain of TIR1. (B) Experimental outline. AID-tagged chromatin remodeler strains were grown to mid-log in YPD. MNase-seq was performed to compare nucleosome positioning before and at two time points after auxin addition. (C) Average MNase coverage positioned relative to the transcription start site (TSS) (“metagene”) for each chromatin remodeler AID strain before and after auxin addition and in the relevant KO strains (if available) (top). Average of the change in MNase coverage before and after auxin addition (1 hr to 0 hr) positioned relative to the TSS for each chromatin remodeler AID strain (bottom). (D) Heatmaps representing the change in MNase coverage before and after auxin addition (1 hr to 0 hr) positioned relative to the TSS (in yellow) for each AID strain. Genes (rows) are sorted, in each strain, by the magnitude of changes in coverage following the depletion in the NFR area. .

    Journal: Cell Reports

    Article Title: Dynamics of Chromatin and Transcription during Transient Depletion of the RSC Chromatin Remodeling Complex

    doi: 10.1016/j.celrep.2018.12.020

    Figure Lengend Snippet: Induced Knockdown Screen of ATP-Dependent Chromatin Remodelers ) yielding an auxin-inducible, rapid degradation of tagged chromatin remodelers. Plant hormone auxin (IAA) directly induces rapid degradation of the AID-tagged protein by mediating the interaction of a degron domain in the target protein with the substrate recognition domain of TIR1. (B) Experimental outline. AID-tagged chromatin remodeler strains were grown to mid-log in YPD. MNase-seq was performed to compare nucleosome positioning before and at two time points after auxin addition. (C) Average MNase coverage positioned relative to the transcription start site (TSS) (“metagene”) for each chromatin remodeler AID strain before and after auxin addition and in the relevant KO strains (if available) (top). Average of the change in MNase coverage before and after auxin addition (1 hr to 0 hr) positioned relative to the TSS for each chromatin remodeler AID strain (bottom). (D) Heatmaps representing the change in MNase coverage before and after auxin addition (1 hr to 0 hr) positioned relative to the TSS (in yellow) for each AID strain. Genes (rows) are sorted, in each strain, by the magnitude of changes in coverage following the depletion in the NFR area. .

    Article Snippet: Spheroplasts were washed, resuspended in NP buffer and treated with MNase (Micrococcal nuclease, Worthington) to generate 80% mono-nucleosomes (1 unit for 2.5 OD initial culture, 37°C, 20 minutes).

    Techniques:

    Bbd-containing nucleosomal arrays repress basal transcription. ( A ) In all, 2.1 μg of assembled Bbd-, mouse- and Drosophila -nucleosomal arrays was digested with 0.12 U MNase. Aliquots of 0.5 μg were removed after 1, 2, 4 and 8 min, deproteinized and analyzed on 1.2% agarose gels. ( B ) In vitro transcription reactions for naked DNA, Bbd-nucleosomal arrays and mouse-nucleosomal arrays are shown. Tax/CREB and p300 were added as indicated above the lanes. Recovery standard and transcript are indicated on the left. ( C ) Results from three transcription assays (from three independent chromatin assembly reactions) were quantified by ImageQuant 5.1 and normalized compared to the recovery standard. Transcripts from mouse and Bbd-nucleosomal arrays are shown by white and gray bars, respectively.

    Journal: The EMBO Journal

    Article Title: Nucleosomes containing the histone variant H2A.Bbd organize only 118 base pairs of DNA

    doi: 10.1038/sj.emboj.7600316

    Figure Lengend Snippet: Bbd-containing nucleosomal arrays repress basal transcription. ( A ) In all, 2.1 μg of assembled Bbd-, mouse- and Drosophila -nucleosomal arrays was digested with 0.12 U MNase. Aliquots of 0.5 μg were removed after 1, 2, 4 and 8 min, deproteinized and analyzed on 1.2% agarose gels. ( B ) In vitro transcription reactions for naked DNA, Bbd-nucleosomal arrays and mouse-nucleosomal arrays are shown. Tax/CREB and p300 were added as indicated above the lanes. Recovery standard and transcript are indicated on the left. ( C ) Results from three transcription assays (from three independent chromatin assembly reactions) were quantified by ImageQuant 5.1 and normalized compared to the recovery standard. Transcripts from mouse and Bbd-nucleosomal arrays are shown by white and gray bars, respectively.

    Article Snippet: Identical amounts of mm -146-NCP and Bbd-146-NCP (2 μg NCP per reaction) were digested with increasing amounts of MNase (Worthington) at 37°C in 125 μl of MNase buffer (0.6 mM HEPES (pH 7.6), 52 mM KCl, 5% (vol/vol) glycerol, 1.4% (wt/vol) polyethylene glycol, 5.4 mM CaCl2 ) for 1.5 min.

    Techniques: In Vitro

    Bbd-NCPs are less resistant against MNase digestion. Identical amounts (2 μg) of mm -146-NCP ( A ) and Bbd-146-NCP ( B ) were digested with increasing amounts of MNase (0, 0.05, 0.1, 0.2, 0.4 U) for 1.5 min. Deproteinized samples were analyzed by 10% PAGE, stained with ethidium bromide. For time courses, 10 μg of mm -196-NCP ( C ) and Bbd-196-NCP ( D ) was incubated with the same amount of MNase (0.15 U) for the indicated times. In all, 2 μg of sample was removed at 0, 2, 4, 8 and 16 min, and the deproteinized samples were analyzed by 10% PAGE. The 146 bp 5sDNA (146), 196 bp 5sDNA (196) and mixtures of 146 and 73 bp α-satellite DNA (146+73) were loaded as controls, as indicated. DNA size markers are given.

    Journal: The EMBO Journal

    Article Title: Nucleosomes containing the histone variant H2A.Bbd organize only 118 base pairs of DNA

    doi: 10.1038/sj.emboj.7600316

    Figure Lengend Snippet: Bbd-NCPs are less resistant against MNase digestion. Identical amounts (2 μg) of mm -146-NCP ( A ) and Bbd-146-NCP ( B ) were digested with increasing amounts of MNase (0, 0.05, 0.1, 0.2, 0.4 U) for 1.5 min. Deproteinized samples were analyzed by 10% PAGE, stained with ethidium bromide. For time courses, 10 μg of mm -196-NCP ( C ) and Bbd-196-NCP ( D ) was incubated with the same amount of MNase (0.15 U) for the indicated times. In all, 2 μg of sample was removed at 0, 2, 4, 8 and 16 min, and the deproteinized samples were analyzed by 10% PAGE. The 146 bp 5sDNA (146), 196 bp 5sDNA (196) and mixtures of 146 and 73 bp α-satellite DNA (146+73) were loaded as controls, as indicated. DNA size markers are given.

    Article Snippet: Identical amounts of mm -146-NCP and Bbd-146-NCP (2 μg NCP per reaction) were digested with increasing amounts of MNase (Worthington) at 37°C in 125 μl of MNase buffer (0.6 mM HEPES (pH 7.6), 52 mM KCl, 5% (vol/vol) glycerol, 1.4% (wt/vol) polyethylene glycol, 5.4 mM CaCl2 ) for 1.5 min.

    Techniques: Polyacrylamide Gel Electrophoresis, Staining, Incubation

    Xla H2A 106 -NCP and Xla H2A 112 -NCP behave like wild-type mm- NCP. ( A ) Salt gradient reconstituted Bbd-NCP (lane 1), Xla -NCP (lanes 6 and 7), Xla H2A 106 -NCP (106NCP, lanes 2 and 3) and Xla H2A 112 -NCP (112NCP, lanes 4 and 5), before (−) and after (+) a 1 h incubation at 37°C, were analyzed by 5% native PAGE, followed by staining with Coomassie blue. ( B–E ) Micrococcal digestion of mutant nucleosomes: 10 μg of Xla -NCP (B), Bbd-NCP (C), Xla H2A 112 -NCP (D) and Xla H2A 106 -NCP (E) were digested with 0.15 U MNase. Aliquots of 2 μg were removed after 0, 2, 4, 8 and 16 min. PAGE (10%) was used to check the deproteinized DNA fragments. 5SDNA (146 bp) (146) was loaded as indicated (D and E, lane 8).

    Journal: The EMBO Journal

    Article Title: Nucleosomes containing the histone variant H2A.Bbd organize only 118 base pairs of DNA

    doi: 10.1038/sj.emboj.7600316

    Figure Lengend Snippet: Xla H2A 106 -NCP and Xla H2A 112 -NCP behave like wild-type mm- NCP. ( A ) Salt gradient reconstituted Bbd-NCP (lane 1), Xla -NCP (lanes 6 and 7), Xla H2A 106 -NCP (106NCP, lanes 2 and 3) and Xla H2A 112 -NCP (112NCP, lanes 4 and 5), before (−) and after (+) a 1 h incubation at 37°C, were analyzed by 5% native PAGE, followed by staining with Coomassie blue. ( B–E ) Micrococcal digestion of mutant nucleosomes: 10 μg of Xla -NCP (B), Bbd-NCP (C), Xla H2A 112 -NCP (D) and Xla H2A 106 -NCP (E) were digested with 0.15 U MNase. Aliquots of 2 μg were removed after 0, 2, 4, 8 and 16 min. PAGE (10%) was used to check the deproteinized DNA fragments. 5SDNA (146 bp) (146) was loaded as indicated (D and E, lane 8).

    Article Snippet: Identical amounts of mm -146-NCP and Bbd-146-NCP (2 μg NCP per reaction) were digested with increasing amounts of MNase (Worthington) at 37°C in 125 μl of MNase buffer (0.6 mM HEPES (pH 7.6), 52 mM KCl, 5% (vol/vol) glycerol, 1.4% (wt/vol) polyethylene glycol, 5.4 mM CaCl2 ) for 1.5 min.

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