mnase  (Worthington Biochemical)


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    Structured Review

    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 .
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    Images

    1) Product Images from "Dynamics of Chromatin and Transcription during Transient Depletion of the RSC Chromatin Remodeling Complex"

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

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2018.12.020

    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 .
    Figure Legend 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 .

    Techniques Used: 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 .
    Figure Legend 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 .

    Techniques Used: 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 .
    Figure Legend 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 .

    Techniques Used: 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 .
    Figure Legend 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 .

    Techniques Used: 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 .
    Figure Legend 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 .

    Techniques Used:

    2) 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:

    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

    3) Product Images from "Enhancer regions show high histone H3.3 turnover that changes during differentiation"

    Article Title: Enhancer regions show high histone H3.3 turnover that changes during differentiation

    Journal: eLife

    doi: 10.7554/eLife.15316

    Measuring DNA accessibility using MNase titration. ( A ) Boxplot of average TI over DNase hypersensitive regions in E14 ESCs (Encode accession ENCSR000CMW). TI index was plotted over called DNase peaks (E14-DS18505.peaks.fdr0.01). ( B ) MNase digestion patterns generated by digestion of ESC and NSC chromatin with varying concentrations of enzyme (1U, 4U, 16U, 64U). ( C ) Sequencing data from each of the above titration points was used to generate an accessibility score (MACC) by fitting these to a linear model. DOI: http://dx.doi.org/10.7554/eLife.15316.019
    Figure Legend Snippet: Measuring DNA accessibility using MNase titration. ( A ) Boxplot of average TI over DNase hypersensitive regions in E14 ESCs (Encode accession ENCSR000CMW). TI index was plotted over called DNase peaks (E14-DS18505.peaks.fdr0.01). ( B ) MNase digestion patterns generated by digestion of ESC and NSC chromatin with varying concentrations of enzyme (1U, 4U, 16U, 64U). ( C ) Sequencing data from each of the above titration points was used to generate an accessibility score (MACC) by fitting these to a linear model. DOI: http://dx.doi.org/10.7554/eLife.15316.019

    Techniques Used: Titration, Generated, Sequencing

    4) Product Images from "Differential Nucleosome Occupancies across Oct4-Sox2 Binding Sites in Murine Embryonic Stem Cells"

    Article Title: Differential Nucleosome Occupancies across Oct4-Sox2 Binding Sites in Murine Embryonic Stem Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0127214

    Mapping in vivo and in vitro nucleosome occupancy using BAC-based enrichment. (a) Protocols were modified so that permeabilization and micrococcal nuclease digestion occur while embryonic stem cells are attached to the tissue culture surface to improve recovery and digestion reproducibility. The amount of micrococcal nuclease (MNase) in the digestion was titrated so that the mononucleosome band at 147bp is the primary band, without overdigestion. Digests were measured in Worthington Units of MNase * Time of digestion / volume of cell culture (U*min/mL). Lanes 1 and 8: 50bp ladder. Lanes 2–7 range from 2500 U*min/mL to 25,000 U*min/mL of MNase, with ideal digestion in the third condition, 10,000 U*min/mL. This amount of digestion was used in all future experiments. (b) Genomic DNA was purified from embryonic stem cell cultures and combined with histone octamer purified from chicken erythrocytes in a ratio of 100μg:30μg under high salt (2M NaCl) conditions. Removal of salt via dialysis results in reconstituted chromatin, representing histone proteins’ preferred DNA sequences. Reconstituted chromatin was digested with 5 Worthington Units of micrococcal nuclease per 10μg of genomic DNA present, for a digestion of 5 minutes at 37°C (Lanes 1 and 7: 50 bp ladder; Lanes 2–6: digested chromatin). (c) Bacterial Artificial Chromosome (BAC) DNA, which was nicked with biotin-dUTP, was blocked with Cot-1 DNA at a ratio of 100ng:10μg. 1μg of library-adapted mononucleosome DNA was denatured and mixed with BAC DNA. Mononucleosome DNA was hybridized to the corresponding BAC region and was isolated by removing BACs from solution with streptavadin beads, stringently washing the beads, and eluting single stranded DNA from the beads. Double stranded products were amplified using PCR and sent for paired-end sequencing.
    Figure Legend Snippet: Mapping in vivo and in vitro nucleosome occupancy using BAC-based enrichment. (a) Protocols were modified so that permeabilization and micrococcal nuclease digestion occur while embryonic stem cells are attached to the tissue culture surface to improve recovery and digestion reproducibility. The amount of micrococcal nuclease (MNase) in the digestion was titrated so that the mononucleosome band at 147bp is the primary band, without overdigestion. Digests were measured in Worthington Units of MNase * Time of digestion / volume of cell culture (U*min/mL). Lanes 1 and 8: 50bp ladder. Lanes 2–7 range from 2500 U*min/mL to 25,000 U*min/mL of MNase, with ideal digestion in the third condition, 10,000 U*min/mL. This amount of digestion was used in all future experiments. (b) Genomic DNA was purified from embryonic stem cell cultures and combined with histone octamer purified from chicken erythrocytes in a ratio of 100μg:30μg under high salt (2M NaCl) conditions. Removal of salt via dialysis results in reconstituted chromatin, representing histone proteins’ preferred DNA sequences. Reconstituted chromatin was digested with 5 Worthington Units of micrococcal nuclease per 10μg of genomic DNA present, for a digestion of 5 minutes at 37°C (Lanes 1 and 7: 50 bp ladder; Lanes 2–6: digested chromatin). (c) Bacterial Artificial Chromosome (BAC) DNA, which was nicked with biotin-dUTP, was blocked with Cot-1 DNA at a ratio of 100ng:10μg. 1μg of library-adapted mononucleosome DNA was denatured and mixed with BAC DNA. Mononucleosome DNA was hybridized to the corresponding BAC region and was isolated by removing BACs from solution with streptavadin beads, stringently washing the beads, and eluting single stranded DNA from the beads. Double stranded products were amplified using PCR and sent for paired-end sequencing.

    Techniques Used: In Vivo, In Vitro, BAC Assay, Modification, Cell Culture, Purification, Isolation, Amplification, Polymerase Chain Reaction, Sequencing

    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:

    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 "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

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    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: Counteracting H3K4 methylation modulators Set1 and Jhd2 co-regulate chromatin dynamics and gene transcription
    Article Snippet: Spheroplasts were washed twice with 25 ml wash buffer (50 mM Tris pH 7.5, 100 mM KCl, 2.5 mM MgCl2 , 400 mM Sorbitol and PIs), resuspended in MNase digestion buffer (50mm Tris pH 7.5, 100 mM NaCl, 5 mM MgCl2 , 1 mM CaCl2 and 0.1% Triton X-100 and PIs) and digested with 10 units of MNase (Micrococcal Nuclease, Worthington Biomedical) at 37 °C for 15 min. Digestion was stopped by adding 100 μl 10 × stop buffer (50 mM Tris–HCl pH 7.5, 500 mM NaCl, 100 mM EDTA, 20 mM EGTA, 10% Triton X-100 and 1% sodium deoxycholate). .. After centrifugation, soluble chromatin (500 μl) was mixed with an equal amount of elution solution (1% SDS, 100 mM sodium bicarbonate) and NaCl (200 mM final concentration), reverse cross-linked for 5 h at 65 °C and ethanol precipitated overnight at −20 °C.

    Blocking Assay:

    Article Title: Counteracting H3K4 methylation modulators Set1 and Jhd2 co-regulate chromatin dynamics and gene transcription
    Article Snippet: Nuclei were washed with micrococcal nuclease (MNase) digestion buffer (25 mM Tris–HCl pH 7.5, 50 mM NaCl, 5 mM MgCl2 , 1 mM CaCl2 ) and resuspended in the same buffer before digesting chromatin with 20 units of MNase (Worthington Biochemicals) for 15 min at 37 °C. .. Nuclei were washed with micrococcal nuclease (MNase) digestion buffer (25 mM Tris–HCl pH 7.5, 50 mM NaCl, 5 mM MgCl2 , 1 mM CaCl2 ) and resuspended in the same buffer before digesting chromatin with 20 units of MNase (Worthington Biochemicals) for 15 min at 37 °C.

    Electrophoresis:

    Article Title: Mitotic post-translational modifications of histones promote chromatin compaction in vitro
    Article Snippet: Reconstituted chromatin was fixed with 0.375% formaldehyde for 15 min at room temperature, before addition of CaCl2 to a final concentration of 5 mM; 1 U of microccocal nuclease (Worthington) was used for each reaction. .. Reconstituted chromatin was fixed with 0.375% formaldehyde for 15 min at room temperature, before addition of CaCl2 to a final concentration of 5 mM; 1 U of microccocal nuclease (Worthington) was used for each reaction.

    Incubation:

    Article Title: Tax Abolishes Histone H1 Repression of p300 Acetyltransferase Activity at the Human T-Cell Leukemia Virus Type 1 Promoter
    Article Snippet: Micrococcal nuclease (MNase) digestion was performed on 2.1 μg of assembled DNA (98 μl assembled chromatin). .. 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. .. After 1, 2, 4, and 8 min, 60 μl aliquots were removed and digestion was stopped with 12 μl Tris-EDTA (6 μl T10 E1 -6 μl 0.5 M EDTA).

    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: Selective suppression of antisense transcription by Set2-mediated H3K36 methylation
    Article Snippet: After > 90% of the cells have spheroplasted, the mixture was pelleted at 7,000 g , and resuspended in 2 ml of NP buffer (1 M Sorbitol, 50 mM NaCl, 10 mM Tris pH 7.4, 5 mM MgCl2, 1 mM CaCl2) with 500 μM spermidine, 1 mM B mercaptoethanol and 0.075% NP-40. .. Three 600 μl aliquots of cells were used to titrate MNase (Worthington, 20 U per μl in 10 mM Tris pH 4.0) amounts and incubated at 37 °C for 20 min. to achieve the formation of 80% of mononucleosomes. .. The reactions were stopped with 1 × STOP buffer (1% SDS and 10 mM EDTA).

    Article Title: Human CHD2 Is a Chromatin Assembly ATPase Regulated by Its Chromo- and DNA-binding Domains
    Article Snippet: Briefly, the histone cores were incubated first with the histone chaperone NAP-1 for 20 min on ice. .. The reaction was allowed to proceed for 1 h at room temperature and then partially digested by adding 2 m m CaCl2 and MNase (Worthington; low concentration = 7 mU μl−1 , high concentration = 28 mU μl−1 ).

    Article Title: A synthetic biology approach to probing nucleosome symmetry
    Article Snippet: 1/10 vol freshly dissolved 11 mg/ml dimethyl suberimidate (Pierce) in E buffer was added, and samples were incubated with rotation at room temperature 90 min. For time point aliquots to be analyzed directly on gels, proteins were precipitated by addition of 1/10 vol 100% TCA. .. For material to be MNase-digested and immunoprecipitated, the crosslinker was quenched by addition of 50 mM Tris-Cl, pH 7.5 and further rotation for 15 min. 10 mM MgCl2 and 1 mM CaCl2 were added and tubes were equilibrated at 37°C for 5 min. 20 μl MNase (Worthington, 20 U/μl in 10 mM Tris-Cl pH 7.4) were added, tubes were inverted five times and incubated at 37°C, 20 min. Digestion was stopped by moving tubes to 4°C, adding 40 μl 0.25 M EDTA/EGTA and inversion to mix. .. Material was centrifuged at 8000 x g for 1 min, 4°C, and pre-cleared by incubation with CL2B-sepharose beads for 30 min 4°C and centrifugation at 8000 x g for 1 min, 4°C.

    Article Title: Mitotic post-translational modifications of histones promote chromatin compaction in vitro
    Article Snippet: Reconstituted chromatin was fixed with 0.375% formaldehyde for 15 min at room temperature, before addition of CaCl2 to a final concentration of 5 mM; 1 U of microccocal nuclease (Worthington) was used for each reaction. .. Reconstituted chromatin was fixed with 0.375% formaldehyde for 15 min at room temperature, before addition of CaCl2 to a final concentration of 5 mM; 1 U of microccocal nuclease (Worthington) was used for each reaction.

    Article Title: Counteracting H3K4 methylation modulators Set1 and Jhd2 co-regulate chromatin dynamics and gene transcription
    Article Snippet: Cross-linked cells were resuspended in 1 ml fresh spheroplasting buffer before adding Lyticase (500 units; Sigma) and incubation in a 37 °C water-bath shaker for 15–20 min with gentle agitation. .. Spheroplasts were washed twice with 25 ml wash buffer (50 mM Tris pH 7.5, 100 mM KCl, 2.5 mM MgCl2 , 400 mM Sorbitol and PIs), resuspended in MNase digestion buffer (50mm Tris pH 7.5, 100 mM NaCl, 5 mM MgCl2 , 1 mM CaCl2 and 0.1% Triton X-100 and PIs) and digested with 10 units of MNase (Micrococcal Nuclease, Worthington Biomedical) at 37 °C for 15 min. Digestion was stopped by adding 100 μl 10 × stop buffer (50 mM Tris–HCl pH 7.5, 500 mM NaCl, 100 mM EDTA, 20 mM EGTA, 10% Triton X-100 and 1% sodium deoxycholate).

    Western Blot:

    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). .. 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).

    Transfection:

    Article Title: Tousled-like kinases stabilize replication forks and show synthetic lethality with checkpoint and PARP inhibitors
    Article Snippet: Thirty hours after transfection, nascent chromatin was labeled with [3 H]thymidine (25 nCi/ml). .. Cells were lysed in hypotonic buffer [10 mM tris (pH 7.4), 2.5 mM MgCl2 , and 0.5% NP-40, protease and phosphatase inhibitors], and nuclei were resuspended in digestion buffer [10 mM tris (pH 7.4), 10 mM NaCl, 5 mM MgCl2 , and 2 mM CaCl2 , protease and phosphatase inhibitors] and subjected to digestion with MNase (0.01 U/μl) (Worthington Biochemical Co.) at 37°C.

    TCA Precipitation:

    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). .. 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).

    Concentration Assay:

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

    Article Title: Differential Nucleosome Occupancies across Oct4-Sox2 Binding Sites in Murine Embryonic Stem Cells
    Article Snippet: Cells were permeablized while adherent to culture dishes with lysis buffer (10mM Tris-Cl (pH 7.4), 10mM NaCl, 3mM MgCl2 , 0.5% NP-40, 0.15mM spermine, 0.5mM spermidine). .. Chromatin was then digested with micrococcal nuclease (Sigma N3755) at a concentration of 1000 Worthington units of MNase per 100 mm culture plate in digestion buffer (10mM Tris-Cl (pH 7.4), 15mM NaCl, 60mM KCl, 0.15mM spermine, 0.5mM spermidine, 1mM CaCl2 ) for 30–60 minutes at room temperature. .. Digestion was stopped with 1/10 volume stop solution (0.25M EDTA, 5% SDS).

    Article Title: Selective suppression of antisense transcription by Set2-mediated H3K36 methylation
    Article Snippet: Zymolase (10 mg ml−1 in buffer Z) was added to a final concentration of 0.5 mg per ml and incubated at 30 °C with continuous mixing in a water bath. .. Three 600 μl aliquots of cells were used to titrate MNase (Worthington, 20 U per μl in 10 mM Tris pH 4.0) amounts and incubated at 37 °C for 20 min. to achieve the formation of 80% of mononucleosomes.

    Article Title: Novel nucleosomal particles containing core histones and linker DNA but no histone H1
    Article Snippet: A 1.4 ml-aliquot of nuclei in a microfuge tube was adjusted to 2 mM CaCl2 ( = 1 mM excess over EDTA), warmed briefly to 37°C, and digested with MNase (20 Worthington units) for 3 min. .. A 1.4 ml-aliquot of nuclei in a microfuge tube was adjusted to 2 mM CaCl2 ( = 1 mM excess over EDTA), warmed briefly to 37°C, and digested with MNase (20 Worthington units) for 3 min.

    Article Title: Human CHD2 Is a Chromatin Assembly ATPase Regulated by Its Chromo- and DNA-binding Domains
    Article Snippet: For minus ATP reactions, only ATP was left out; all other components of the regeneration system were included. .. The reaction was allowed to proceed for 1 h at room temperature and then partially digested by adding 2 m m CaCl2 and MNase (Worthington; low concentration = 7 mU μl−1 , high concentration = 28 mU μl−1 ). .. After 4 min at room temperature, the reactions were stopped with the addition of 125 μl of Stop Buffer (1% ( w / v ) SDS, 200 m m NaCl, 250 μg/ml glycogen, 20 m m EDTA pH 8.0) and proteinase K (Worthington) at a final concentration of 50 μg/ml.

    Article Title: Mitotic post-translational modifications of histones promote chromatin compaction in vitro
    Article Snippet: The digestion products were visualized by electrophoresis on a 5% acrylamide (37.5 : 1) gel in 0.2× TBE buffer. .. Reconstituted chromatin was fixed with 0.375% formaldehyde for 15 min at room temperature, before addition of CaCl2 to a final concentration of 5 mM; 1 U of microccocal nuclease (Worthington) was used for each reaction. .. Digestion was performed on ice in a buffer containing 50 mM Tris–Cl pH 7.6, 5 mM CaCl2 , 0.5 mM PMSF, for the indicated time periods, and the reaction was stopped by the addition of excess EGTA.

    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).

    Cell Culture:

    Article Title: Downstream Antisense Transcription Predicts Genomic Features That Define the Specific Chromatin Environment at Mammalian Promoters
    Article Snippet: The list of genes with T47D/A1-2-specific daTSSs was applied to Ingenuity Pathway Analysis [ ] considering experimentally observed associations over mammalian tissues and cell lines. .. Nuclei were harvested from cultured T47D/A1-2 cells and digested for 5 minutes at 37°C with a range of MNase (Worthington) concentrations. .. Reactions were stopped by the addition of EDTA and then treated with RNase and proteinase K. Digested DNA was isolated by phenol/chloroform extraction and ethanol precipitation.

    Generated:

    Article Title: Downstream Antisense Transcription Predicts Genomic Features That Define the Specific Chromatin Environment at Mammalian Promoters
    Article Snippet: Nuclei were harvested from cultured T47D/A1-2 cells and digested for 5 minutes at 37°C with a range of MNase (Worthington) concentrations. .. Libraries were prepared using an Illumina TruSeq sample preparation kit and sequenced on an Illumina HiSeq for paired-end 50 base reads.

    DNA Sequencing:

    Article Title: Dynamics of Chromatin and Transcription during Transient Depletion of the RSC Chromatin Remodeling Complex
    Article Snippet: Paragraph title: MNase digestion and DNA Sequencing ... 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).

    Polymerase Chain Reaction:

    Article Title: Counteracting H3K4 methylation modulators Set1 and Jhd2 co-regulate chromatin dynamics and gene transcription
    Article Snippet: Spheroplasts were washed twice with 25 ml wash buffer (50 mM Tris pH 7.5, 100 mM KCl, 2.5 mM MgCl2 , 400 mM Sorbitol and PIs), resuspended in MNase digestion buffer (50mm Tris pH 7.5, 100 mM NaCl, 5 mM MgCl2 , 1 mM CaCl2 and 0.1% Triton X-100 and PIs) and digested with 10 units of MNase (Micrococcal Nuclease, Worthington Biomedical) at 37 °C for 15 min. Digestion was stopped by adding 100 μl 10 × stop buffer (50 mM Tris–HCl pH 7.5, 500 mM NaCl, 100 mM EDTA, 20 mM EGTA, 10% Triton X-100 and 1% sodium deoxycholate). .. Spheroplasts were washed twice with 25 ml wash buffer (50 mM Tris pH 7.5, 100 mM KCl, 2.5 mM MgCl2 , 400 mM Sorbitol and PIs), resuspended in MNase digestion buffer (50mm Tris pH 7.5, 100 mM NaCl, 5 mM MgCl2 , 1 mM CaCl2 and 0.1% Triton X-100 and PIs) and digested with 10 units of MNase (Micrococcal Nuclease, Worthington Biomedical) at 37 °C for 15 min. Digestion was stopped by adding 100 μl 10 × stop buffer (50 mM Tris–HCl pH 7.5, 500 mM NaCl, 100 mM EDTA, 20 mM EGTA, 10% Triton X-100 and 1% sodium deoxycholate).

    Recombinant:

    Article Title: Human CHD2 Is a Chromatin Assembly ATPase Regulated by Its Chromo- and DNA-binding Domains
    Article Snippet: A standard 70 μl assembly reaction contained purified core histones (1.4 μg), plasmid DNA (1.4 μg; 3.2 kilobases (kb); pGIE-0), Topo I, NAP-1 (13 μg), recombinant human CHD2 (100 n m final concentration), and an ATP regeneration system (3 m m ATP, 5 m m MgCl2 , 30 m m phosphocreatine, and 5 ng/μl creatine phosphokinase). .. The reaction was allowed to proceed for 1 h at room temperature and then partially digested by adding 2 m m CaCl2 and MNase (Worthington; low concentration = 7 mU μl−1 , high concentration = 28 mU μl−1 ).

    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).

    Article Title: Counteracting H3K4 methylation modulators Set1 and Jhd2 co-regulate chromatin dynamics and gene transcription
    Article Snippet: Paragraph title: ChIP-seq ... Nuclei were washed with micrococcal nuclease (MNase) digestion buffer (25 mM Tris–HCl pH 7.5, 50 mM NaCl, 5 mM MgCl2 , 1 mM CaCl2 ) and resuspended in the same buffer before digesting chromatin with 20 units of MNase (Worthington Biochemicals) for 15 min at 37 °C.

    In Vivo:

    Article Title: Differential Nucleosome Occupancies across Oct4-Sox2 Binding Sites in Murine Embryonic Stem Cells
    Article Snippet: Paragraph title: In vivo mononucleosome purification ... Chromatin was then digested with micrococcal nuclease (Sigma N3755) at a concentration of 1000 Worthington units of MNase per 100 mm culture plate in digestion buffer (10mM Tris-Cl (pH 7.4), 15mM NaCl, 60mM KCl, 0.15mM spermine, 0.5mM spermidine, 1mM CaCl2 ) for 30–60 minutes at room temperature.

    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 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.

    Magnetic Beads:

    Article Title: Counteracting H3K4 methylation modulators Set1 and Jhd2 co-regulate chromatin dynamics and gene transcription
    Article Snippet: Nuclei were washed with micrococcal nuclease (MNase) digestion buffer (25 mM Tris–HCl pH 7.5, 50 mM NaCl, 5 mM MgCl2 , 1 mM CaCl2 ) and resuspended in the same buffer before digesting chromatin with 20 units of MNase (Worthington Biochemicals) for 15 min at 37 °C. .. Nuclei were washed with micrococcal nuclease (MNase) digestion buffer (25 mM Tris–HCl pH 7.5, 50 mM NaCl, 5 mM MgCl2 , 1 mM CaCl2 ) and resuspended in the same buffer before digesting chromatin with 20 units of MNase (Worthington Biochemicals) for 15 min at 37 °C.

    Mutagenesis:

    Article Title: Selective suppression of antisense transcription by Set2-mediated H3K36 methylation
    Article Snippet: Wild-type and set2 mutant were grown in 200 ml of YPD, crosslinked and treated with zymolase to spheroplast the yeast cells. .. Three 600 μl aliquots of cells were used to titrate MNase (Worthington, 20 U per μl in 10 mM Tris pH 4.0) amounts and incubated at 37 °C for 20 min. to achieve the formation of 80% of mononucleosomes.

    Isolation:

    Article Title: Hierarchical regulation of the genome: global changes in nucleosome organization potentiate genome response
    Article Snippet: 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.

    Article Title: Coordinated repressive chromatin-remodeling of Oct4 and Nanog genes in RA-induced differentiation of embryonic stem cells involves RIP140
    Article Snippet: Micrococcal nuclease (MNase) digestion was performed as described ( ). .. 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: Differential Nucleosome Occupancies across Oct4-Sox2 Binding Sites in Murine Embryonic Stem Cells
    Article Snippet: Chromatin was then digested with micrococcal nuclease (Sigma N3755) at a concentration of 1000 Worthington units of MNase per 100 mm culture plate in digestion buffer (10mM Tris-Cl (pH 7.4), 15mM NaCl, 60mM KCl, 0.15mM spermine, 0.5mM spermidine, 1mM CaCl2 ) for 30–60 minutes at room temperature. .. Chromatin was then digested with micrococcal nuclease (Sigma N3755) at a concentration of 1000 Worthington units of MNase per 100 mm culture plate in digestion buffer (10mM Tris-Cl (pH 7.4), 15mM NaCl, 60mM KCl, 0.15mM spermine, 0.5mM spermidine, 1mM CaCl2 ) for 30–60 minutes at room temperature.

    Article Title: Promyelocytic leukemia protein in retinoic acid-induced chromatin remodeling of Oct4 gene promoter
    Article Snippet: Micrococcal nuclease (MNase) digestion was performed as described [ ]. .. 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: 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.

    Labeling:

    Article Title: Tousled-like kinases stabilize replication forks and show synthetic lethality with checkpoint and PARP inhibitors
    Article Snippet: To compensate for lower replication rate in TLK2- and FLASH-depleted cells, labeling times were adjusted to obtain similar [3 H]thymidine incorporation (10, 15, and 30 min for siRNA control, siTLK2, and siFLASH, respectively). .. Cells were lysed in hypotonic buffer [10 mM tris (pH 7.4), 2.5 mM MgCl2 , and 0.5% NP-40, protease and phosphatase inhibitors], and nuclei were resuspended in digestion buffer [10 mM tris (pH 7.4), 10 mM NaCl, 5 mM MgCl2 , and 2 mM CaCl2 , protease and phosphatase inhibitors] and subjected to digestion with MNase (0.01 U/μl) (Worthington Biochemical Co.) at 37°C.

    Purification:

    Article Title: Hierarchical regulation of the genome: global changes in nucleosome organization potentiate genome response
    Article Snippet: Paragraph title: 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: 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. .. 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.

    Article Title: Differential Nucleosome Occupancies across Oct4-Sox2 Binding Sites in Murine Embryonic Stem Cells
    Article Snippet: Paragraph title: In vivo mononucleosome purification ... Chromatin was then digested with micrococcal nuclease (Sigma N3755) at a concentration of 1000 Worthington units of MNase per 100 mm culture plate in digestion buffer (10mM Tris-Cl (pH 7.4), 15mM NaCl, 60mM KCl, 0.15mM spermine, 0.5mM spermidine, 1mM CaCl2 ) for 30–60 minutes at room temperature.

    Article Title: Enhancer regions show high histone H3.3 turnover that changes during differentiation
    Article Snippet: Digestions were performed on 2.5 105 nuclei per reaction using 64U, 16U, 4U or 1U MNase (Worthington) in 10 mM Tris pH 7.4, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2 for 10–15 min at 25°C. .. Digestions were performed on 2.5 105 nuclei per reaction using 64U, 16U, 4U or 1U MNase (Worthington) in 10 mM Tris pH 7.4, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2 for 10–15 min at 25°C.

    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: Sucrose density gradient experiments were performed as described previously [ ]. .. 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). .. After microcentrifugation at 5,000 rpm for 5 min, the nuclear pellet was resuspended in 0.65 ml of the elution buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl) containing 5 mM EDTA/EGTA, gently rocked for 1 hr at 4°C followed by microcentrifugation to obtain soluble nucleosomes.

    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. .. 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.

    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 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: Selective suppression of antisense transcription by Set2-mediated H3K36 methylation
    Article Snippet: Three 600 μl aliquots of cells were used to titrate MNase (Worthington, 20 U per μl in 10 mM Tris pH 4.0) amounts and incubated at 37 °C for 20 min. to achieve the formation of 80% of mononucleosomes. .. Three 600 μl aliquots of cells were used to titrate MNase (Worthington, 20 U per μl in 10 mM Tris pH 4.0) amounts and incubated at 37 °C for 20 min. to achieve the formation of 80% of mononucleosomes.

    Article Title: Human CHD2 Is a Chromatin Assembly ATPase Regulated by Its Chromo- and DNA-binding Domains
    Article Snippet: A standard 70 μl assembly reaction contained purified core histones (1.4 μg), plasmid DNA (1.4 μg; 3.2 kilobases (kb); pGIE-0), Topo I, NAP-1 (13 μg), recombinant human CHD2 (100 n m final concentration), and an ATP regeneration system (3 m m ATP, 5 m m MgCl2 , 30 m m phosphocreatine, and 5 ng/μl creatine phosphokinase). .. The reaction was allowed to proceed for 1 h at room temperature and then partially digested by adding 2 m m CaCl2 and MNase (Worthington; low concentration = 7 mU μl−1 , high concentration = 28 mU μl−1 ).

    Article Title: Counteracting H3K4 methylation modulators Set1 and Jhd2 co-regulate chromatin dynamics and gene transcription
    Article Snippet: Spheroplasts were washed twice with 25 ml wash buffer (50 mM Tris pH 7.5, 100 mM KCl, 2.5 mM MgCl2 , 400 mM Sorbitol and PIs), resuspended in MNase digestion buffer (50mm Tris pH 7.5, 100 mM NaCl, 5 mM MgCl2 , 1 mM CaCl2 and 0.1% Triton X-100 and PIs) and digested with 10 units of MNase (Micrococcal Nuclease, Worthington Biomedical) at 37 °C for 15 min. Digestion was stopped by adding 100 μl 10 × stop buffer (50 mM Tris–HCl pH 7.5, 500 mM NaCl, 100 mM EDTA, 20 mM EGTA, 10% Triton X-100 and 1% sodium deoxycholate). .. Spheroplasts were washed twice with 25 ml wash buffer (50 mM Tris pH 7.5, 100 mM KCl, 2.5 mM MgCl2 , 400 mM Sorbitol and PIs), resuspended in MNase digestion buffer (50mm Tris pH 7.5, 100 mM NaCl, 5 mM MgCl2 , 1 mM CaCl2 and 0.1% Triton X-100 and PIs) and digested with 10 units of MNase (Micrococcal Nuclease, Worthington Biomedical) at 37 °C for 15 min. Digestion was stopped by adding 100 μl 10 × stop buffer (50 mM Tris–HCl pH 7.5, 500 mM NaCl, 100 mM EDTA, 20 mM EGTA, 10% Triton X-100 and 1% sodium deoxycholate).

    Sequencing:

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

    Lysis:

    Article Title: Differential Nucleosome Occupancies across Oct4-Sox2 Binding Sites in Murine Embryonic Stem Cells
    Article Snippet: Cells were permeablized while adherent to culture dishes with lysis buffer (10mM Tris-Cl (pH 7.4), 10mM NaCl, 3mM MgCl2 , 0.5% NP-40, 0.15mM spermine, 0.5mM spermidine). .. Chromatin was then digested with micrococcal nuclease (Sigma N3755) at a concentration of 1000 Worthington units of MNase per 100 mm culture plate in digestion buffer (10mM Tris-Cl (pH 7.4), 15mM NaCl, 60mM KCl, 0.15mM spermine, 0.5mM spermidine, 1mM CaCl2 ) for 30–60 minutes at room temperature.

    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: 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: 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).

    Agarose Gel Electrophoresis:

    Article Title: Hierarchical regulation of the genome: global changes in nucleosome organization potentiate genome response
    Article Snippet: 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). .. 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: 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.

    Article Title: Human CHD2 Is a Chromatin Assembly ATPase Regulated by Its Chromo- and DNA-binding Domains
    Article Snippet: The reaction was allowed to proceed for 1 h at room temperature and then partially digested by adding 2 m m CaCl2 and MNase (Worthington; low concentration = 7 mU μl−1 , high concentration = 28 mU μl−1 ). .. After 4 min at room temperature, the reactions were stopped with the addition of 125 μl of Stop Buffer (1% ( w / v ) SDS, 200 m m NaCl, 250 μg/ml glycogen, 20 m m EDTA pH 8.0) and proteinase K (Worthington) at a final concentration of 50 μg/ml.

    Article Title: Mitotic post-translational modifications of histones promote chromatin compaction in vitro
    Article Snippet: Reconstituted chromatin was fixed with 0.375% formaldehyde for 15 min at room temperature, before addition of CaCl2 to a final concentration of 5 mM; 1 U of microccocal nuclease (Worthington) was used for each reaction. .. Reconstituted chromatin was fixed with 0.375% formaldehyde for 15 min at room temperature, before addition of CaCl2 to a final concentration of 5 mM; 1 U of microccocal nuclease (Worthington) was used for each reaction.

    Titration:

    Article Title: Hierarchical regulation of the genome: global changes in nucleosome organization potentiate genome response
    Article Snippet: Next, the nuclei were isolated in nucleus isolation buffer containing: 10 mM HEPES at pH 7.8, 2 mM MgOAc2 , 0.3 M sucrose, 1 mM CaCl2 , and 1% Nonidet P-40. .. 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 MNase digestion reactions were stopped with 50 mM EDTA.

    Article Title: Enhancer regions show high histone H3.3 turnover that changes during differentiation
    Article Snippet: Paragraph title: MNase titration ... Digestions were performed on 2.5 105 nuclei per reaction using 64U, 16U, 4U or 1U MNase (Worthington) in 10 mM Tris pH 7.4, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2 for 10–15 min at 25°C.

    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).

    Plasmid Preparation:

    Article Title: Human CHD2 Is a Chromatin Assembly ATPase Regulated by Its Chromo- and DNA-binding Domains
    Article Snippet: After 20 min, the ATP regeneration system, relaxed plasmid DNA (with Topo I still present), and CHD2 were added to the histone/chaperone mixture. .. The reaction was allowed to proceed for 1 h at room temperature and then partially digested by adding 2 m m CaCl2 and MNase (Worthington; low concentration = 7 mU μl−1 , high concentration = 28 mU μl−1 ).

    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).

    Sample Prep:

    Article Title: Downstream Antisense Transcription Predicts Genomic Features That Define the Specific Chromatin Environment at Mammalian Promoters
    Article Snippet: Nuclei were harvested from cultured T47D/A1-2 cells and digested for 5 minutes at 37°C with a range of MNase (Worthington) concentrations. .. Reactions were stopped by the addition of EDTA and then treated with RNase and proteinase K. Digested DNA was isolated by phenol/chloroform extraction and ethanol precipitation.

    Ethanol Precipitation:

    Article Title: Differential Nucleosome Occupancies across Oct4-Sox2 Binding Sites in Murine Embryonic Stem Cells
    Article Snippet: Chromatin was then digested with micrococcal nuclease (Sigma N3755) at a concentration of 1000 Worthington units of MNase per 100 mm culture plate in digestion buffer (10mM Tris-Cl (pH 7.4), 15mM NaCl, 60mM KCl, 0.15mM spermine, 0.5mM spermidine, 1mM CaCl2 ) for 30–60 minutes at room temperature. .. Chromatin was then digested with micrococcal nuclease (Sigma N3755) at a concentration of 1000 Worthington units of MNase per 100 mm culture plate in digestion buffer (10mM Tris-Cl (pH 7.4), 15mM NaCl, 60mM KCl, 0.15mM spermine, 0.5mM spermidine, 1mM CaCl2 ) for 30–60 minutes 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. .. 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.

    Article Title: Selective suppression of antisense transcription by Set2-mediated H3K36 methylation
    Article Snippet: Three 600 μl aliquots of cells were used to titrate MNase (Worthington, 20 U per μl in 10 mM Tris pH 4.0) amounts and incubated at 37 °C for 20 min. to achieve the formation of 80% of mononucleosomes. .. Three 600 μl aliquots of cells were used to titrate MNase (Worthington, 20 U per μl in 10 mM Tris pH 4.0) amounts and incubated at 37 °C for 20 min. to achieve the formation of 80% of mononucleosomes.

    Immunoprecipitation:

    Article Title: A synthetic biology approach to probing nucleosome symmetry
    Article Snippet: 1/10 vol freshly dissolved 11 mg/ml dimethyl suberimidate (Pierce) in E buffer was added, and samples were incubated with rotation at room temperature 90 min. For time point aliquots to be analyzed directly on gels, proteins were precipitated by addition of 1/10 vol 100% TCA. .. For material to be MNase-digested and immunoprecipitated, the crosslinker was quenched by addition of 50 mM Tris-Cl, pH 7.5 and further rotation for 15 min. 10 mM MgCl2 and 1 mM CaCl2 were added and tubes were equilibrated at 37°C for 5 min. 20 μl MNase (Worthington, 20 U/μl in 10 mM Tris-Cl pH 7.4) were added, tubes were inverted five times and incubated at 37°C, 20 min. Digestion was stopped by moving tubes to 4°C, adding 40 μl 0.25 M EDTA/EGTA and inversion to mix. .. Material was centrifuged at 8000 x g for 1 min, 4°C, and pre-cleared by incubation with CL2B-sepharose beads for 30 min 4°C and centrifugation at 8000 x g for 1 min, 4°C.

    Marker:

    Article Title: Human CHD2 Is a Chromatin Assembly ATPase Regulated by Its Chromo- and DNA-binding Domains
    Article Snippet: The reaction was allowed to proceed for 1 h at room temperature and then partially digested by adding 2 m m CaCl2 and MNase (Worthington; low concentration = 7 mU μl−1 , high concentration = 28 mU μl−1 ). .. The DNA was then extracted with phenol:chloroform, EtOH precipitated, resolved by agarose gel eletrophoresis, and visualized by ethidium bromide (EtBr) staining.

    Staining:

    Article Title: Human CHD2 Is a Chromatin Assembly ATPase Regulated by Its Chromo- and DNA-binding Domains
    Article Snippet: The reaction was allowed to proceed for 1 h at room temperature and then partially digested by adding 2 m m CaCl2 and MNase (Worthington; low concentration = 7 mU μl−1 , high concentration = 28 mU μl−1 ). .. After 4 min at room temperature, the reactions were stopped with the addition of 125 μl of Stop Buffer (1% ( w / v ) SDS, 200 m m NaCl, 250 μg/ml glycogen, 20 m m EDTA pH 8.0) and proteinase K (Worthington) at a final concentration of 50 μg/ml.

    Article Title: Mitotic post-translational modifications of histones promote chromatin compaction in vitro
    Article Snippet: Reconstituted chromatin was fixed with 0.375% formaldehyde for 15 min at room temperature, before addition of CaCl2 to a final concentration of 5 mM; 1 U of microccocal nuclease (Worthington) was used for each reaction. .. Reconstituted chromatin was fixed with 0.375% formaldehyde for 15 min at room temperature, before addition of CaCl2 to a final concentration of 5 mM; 1 U of microccocal nuclease (Worthington) was used for each reaction.

    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: 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 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.

    Fluorescence In Situ Hybridization:

    Article Title: Counteracting H3K4 methylation modulators Set1 and Jhd2 co-regulate chromatin dynamics and gene transcription
    Article Snippet: Nuclei were washed with micrococcal nuclease (MNase) digestion buffer (25 mM Tris–HCl pH 7.5, 50 mM NaCl, 5 mM MgCl2 , 1 mM CaCl2 ) and resuspended in the same buffer before digesting chromatin with 20 units of MNase (Worthington Biochemicals) for 15 min at 37 °C. .. Nuclei were washed with micrococcal nuclease (MNase) digestion buffer (25 mM Tris–HCl pH 7.5, 50 mM NaCl, 5 mM MgCl2 , 1 mM CaCl2 ) and resuspended in the same buffer before digesting chromatin with 20 units of MNase (Worthington Biochemicals) for 15 min at 37 °C.

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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 .
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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:

    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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gku092

    Figure Lengend 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.

    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.

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

    Mapping in vivo and in vitro nucleosome occupancy using BAC-based enrichment. (a) Protocols were modified so that permeabilization and micrococcal nuclease digestion occur while embryonic stem cells are attached to the tissue culture surface to improve recovery and digestion reproducibility. The amount of micrococcal nuclease (MNase) in the digestion was titrated so that the mononucleosome band at 147bp is the primary band, without overdigestion. Digests were measured in Worthington Units of MNase * Time of digestion / volume of cell culture (U*min/mL). Lanes 1 and 8: 50bp ladder. Lanes 2–7 range from 2500 U*min/mL to 25,000 U*min/mL of MNase, with ideal digestion in the third condition, 10,000 U*min/mL. This amount of digestion was used in all future experiments. (b) Genomic DNA was purified from embryonic stem cell cultures and combined with histone octamer purified from chicken erythrocytes in a ratio of 100μg:30μg under high salt (2M NaCl) conditions. Removal of salt via dialysis results in reconstituted chromatin, representing histone proteins’ preferred DNA sequences. Reconstituted chromatin was digested with 5 Worthington Units of micrococcal nuclease per 10μg of genomic DNA present, for a digestion of 5 minutes at 37°C (Lanes 1 and 7: 50 bp ladder; Lanes 2–6: digested chromatin). (c) Bacterial Artificial Chromosome (BAC) DNA, which was nicked with biotin-dUTP, was blocked with Cot-1 DNA at a ratio of 100ng:10μg. 1μg of library-adapted mononucleosome DNA was denatured and mixed with BAC DNA. Mononucleosome DNA was hybridized to the corresponding BAC region and was isolated by removing BACs from solution with streptavadin beads, stringently washing the beads, and eluting single stranded DNA from the beads. Double stranded products were amplified using PCR and sent for paired-end sequencing.

    Journal: PLoS ONE

    Article Title: Differential Nucleosome Occupancies across Oct4-Sox2 Binding Sites in Murine Embryonic Stem Cells

    doi: 10.1371/journal.pone.0127214

    Figure Lengend Snippet: Mapping in vivo and in vitro nucleosome occupancy using BAC-based enrichment. (a) Protocols were modified so that permeabilization and micrococcal nuclease digestion occur while embryonic stem cells are attached to the tissue culture surface to improve recovery and digestion reproducibility. The amount of micrococcal nuclease (MNase) in the digestion was titrated so that the mononucleosome band at 147bp is the primary band, without overdigestion. Digests were measured in Worthington Units of MNase * Time of digestion / volume of cell culture (U*min/mL). Lanes 1 and 8: 50bp ladder. Lanes 2–7 range from 2500 U*min/mL to 25,000 U*min/mL of MNase, with ideal digestion in the third condition, 10,000 U*min/mL. This amount of digestion was used in all future experiments. (b) Genomic DNA was purified from embryonic stem cell cultures and combined with histone octamer purified from chicken erythrocytes in a ratio of 100μg:30μg under high salt (2M NaCl) conditions. Removal of salt via dialysis results in reconstituted chromatin, representing histone proteins’ preferred DNA sequences. Reconstituted chromatin was digested with 5 Worthington Units of micrococcal nuclease per 10μg of genomic DNA present, for a digestion of 5 minutes at 37°C (Lanes 1 and 7: 50 bp ladder; Lanes 2–6: digested chromatin). (c) Bacterial Artificial Chromosome (BAC) DNA, which was nicked with biotin-dUTP, was blocked with Cot-1 DNA at a ratio of 100ng:10μg. 1μg of library-adapted mononucleosome DNA was denatured and mixed with BAC DNA. Mononucleosome DNA was hybridized to the corresponding BAC region and was isolated by removing BACs from solution with streptavadin beads, stringently washing the beads, and eluting single stranded DNA from the beads. Double stranded products were amplified using PCR and sent for paired-end sequencing.

    Article Snippet: Chromatin was then digested with micrococcal nuclease (Sigma N3755) at a concentration of 1000 Worthington units of MNase per 100 mm culture plate in digestion buffer (10mM Tris-Cl (pH 7.4), 15mM NaCl, 60mM KCl, 0.15mM spermine, 0.5mM spermidine, 1mM CaCl2 ) for 30–60 minutes at room temperature.

    Techniques: In Vivo, In Vitro, BAC Assay, Modification, Cell Culture, Purification, Isolation, Amplification, Polymerase Chain Reaction, Sequencing