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 .
    Mnase, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 93/100, based on 104 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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 "Increasing phosphoproteomic coverage through sequential digestion by complementary proteases"

    Article Title: Increasing phosphoproteomic coverage through sequential digestion by complementary proteases

    Journal: Analytical and bioanalytical chemistry

    doi: 10.1007/s00216-011-5466-5

    Phosphorylation sites identified by each approach. (a) Total number of distinct phosphorylation sites by method. Each experimental approach identified a larger number of phosphorylation sites than Glu-C only workflows. (b) Comparison of unique phosphorylation
    Figure Legend Snippet: Phosphorylation sites identified by each approach. (a) Total number of distinct phosphorylation sites by method. Each experimental approach identified a larger number of phosphorylation sites than Glu-C only workflows. (b) Comparison of unique phosphorylation

    Techniques Used:

    Experimental design and analysis of sequential digestion workflows. (a) Schematic diagram of the three sequential digestion approaches. GSPT closely resembles the traditional workflow with substitution of Glu-C for initial digestion and trypsin digestion
    Figure Legend Snippet: Experimental design and analysis of sequential digestion workflows. (a) Schematic diagram of the three sequential digestion approaches. GSPT closely resembles the traditional workflow with substitution of Glu-C for initial digestion and trypsin digestion

    Techniques Used:

    Evaluation of a Glu-C only based phosphoproteomic workflow. (a) In the traditional phosphoproteomic workflow, proteins are obtained from cell lysates and proteolytically digested with trypsin. The resultant peptides are separated into fractions via strong
    Figure Legend Snippet: Evaluation of a Glu-C only based phosphoproteomic workflow. (a) In the traditional phosphoproteomic workflow, proteins are obtained from cell lysates and proteolytically digested with trypsin. The resultant peptides are separated into fractions via strong

    Techniques Used:

    3) Product Images from "Increasing phosphoproteomic coverage through sequential digestion by complementary proteases"

    Article Title: Increasing phosphoproteomic coverage through sequential digestion by complementary proteases

    Journal: Analytical and bioanalytical chemistry

    doi: 10.1007/s00216-011-5466-5

    Phosphorylation sites identified by each approach. (a) Total number of distinct phosphorylation sites by method. Each experimental approach identified a larger number of phosphorylation sites than Glu-C only workflows. (b) Comparison of unique phosphorylation
    Figure Legend Snippet: Phosphorylation sites identified by each approach. (a) Total number of distinct phosphorylation sites by method. Each experimental approach identified a larger number of phosphorylation sites than Glu-C only workflows. (b) Comparison of unique phosphorylation

    Techniques Used:

    Experimental design and analysis of sequential digestion workflows. (a) Schematic diagram of the three sequential digestion approaches. GSPT closely resembles the traditional workflow with substitution of Glu-C for initial digestion and trypsin digestion
    Figure Legend Snippet: Experimental design and analysis of sequential digestion workflows. (a) Schematic diagram of the three sequential digestion approaches. GSPT closely resembles the traditional workflow with substitution of Glu-C for initial digestion and trypsin digestion

    Techniques Used:

    Evaluation of a Glu-C only based phosphoproteomic workflow. (a) In the traditional phosphoproteomic workflow, proteins are obtained from cell lysates and proteolytically digested with trypsin. The resultant peptides are separated into fractions via strong
    Figure Legend Snippet: Evaluation of a Glu-C only based phosphoproteomic workflow. (a) In the traditional phosphoproteomic workflow, proteins are obtained from cell lysates and proteolytically digested with trypsin. The resultant peptides are separated into fractions via strong

    Techniques Used:

    4) Product Images from "Reversible histone glycation is associated with disease-related changes in chromatin architecture"

    Article Title: Reversible histone glycation is associated with disease-related changes in chromatin architecture

    Journal: Nature Communications

    doi: 10.1038/s41467-019-09192-z

    Histone glycation changes chromatin architecture in vitro and in cellulo. a Reconstituted nucleosomal 12mer arrays were incubated with 1 mM MGO for 12 h and analyzed by APAGE (agarose polyacrylamide native gel), either with EtBr or western blot utilizing the indicated antibodies to show the stability of the arrays upon MGO treatment. b Nucleosomal arrays were treated with 100 mM MGO (corresponding to 1:30 sites:MGO stoichiometry) for 12 h at 4 °C. Left: Representative single-molecule unfolding trajectories of MGO-treated (red) and untreated (blue) array obtained by an optical tweezer experiment. Right: Histograms of forces required to unravel each nucleosome in the MGO-treated nucleosomal array (red) and untreated (blue) array. Statistical analysis was based on sampling more than 50 single-molecule data and performing a t test. c Left: magnesium compaction assay of nucleosomal arrays treated with different concentrations of MGO. (+) 10 mM (corresponding to 1:3 sites:MGO stoichiometry); (+++) 100 mM MGO (corresponding to 1:30 sites:MGO stoichiometry). Right: array treated with low MGO concentration for different time points. (+) 2 mM MGO (corresponding to 1:0.6 sites:MGO stoichiometry). Error bars represent standard deviation from three different experiments. d MNase digestion of nucleosomal arrays treated with increasing amounts of MGO. Samples were separated on a native gel and stained with EtBr. e ATAC-seq performed on 293T cells either untreated or treated with 0.5 mM MGO for 12 h. Heatmaps showing the density of mapped ATAC-seq reads at consensus nucleosome peaks and 1000 bp up and downstream from the nucleosome dyad. Line graphs above show the same data as the mean read depth over all nucleosome peaks. f Line graph of the sum of ATAC-seq reads over RefSeq annotated transcriptional start sites show a decrease in chromatin accessibility in MGO-treated cells. g Schematic representation of the results presented in ( b – d )
    Figure Legend Snippet: Histone glycation changes chromatin architecture in vitro and in cellulo. a Reconstituted nucleosomal 12mer arrays were incubated with 1 mM MGO for 12 h and analyzed by APAGE (agarose polyacrylamide native gel), either with EtBr or western blot utilizing the indicated antibodies to show the stability of the arrays upon MGO treatment. b Nucleosomal arrays were treated with 100 mM MGO (corresponding to 1:30 sites:MGO stoichiometry) for 12 h at 4 °C. Left: Representative single-molecule unfolding trajectories of MGO-treated (red) and untreated (blue) array obtained by an optical tweezer experiment. Right: Histograms of forces required to unravel each nucleosome in the MGO-treated nucleosomal array (red) and untreated (blue) array. Statistical analysis was based on sampling more than 50 single-molecule data and performing a t test. c Left: magnesium compaction assay of nucleosomal arrays treated with different concentrations of MGO. (+) 10 mM (corresponding to 1:3 sites:MGO stoichiometry); (+++) 100 mM MGO (corresponding to 1:30 sites:MGO stoichiometry). Right: array treated with low MGO concentration for different time points. (+) 2 mM MGO (corresponding to 1:0.6 sites:MGO stoichiometry). Error bars represent standard deviation from three different experiments. d MNase digestion of nucleosomal arrays treated with increasing amounts of MGO. Samples were separated on a native gel and stained with EtBr. e ATAC-seq performed on 293T cells either untreated or treated with 0.5 mM MGO for 12 h. Heatmaps showing the density of mapped ATAC-seq reads at consensus nucleosome peaks and 1000 bp up and downstream from the nucleosome dyad. Line graphs above show the same data as the mean read depth over all nucleosome peaks. f Line graph of the sum of ATAC-seq reads over RefSeq annotated transcriptional start sites show a decrease in chromatin accessibility in MGO-treated cells. g Schematic representation of the results presented in ( b – d )

    Techniques Used: In Vitro, Incubation, Western Blot, Sampling, Concentration Assay, Standard Deviation, Staining

    5) Product Images from "Increasing phosphoproteomic coverage through sequential digestion by complementary proteases"

    Article Title: Increasing phosphoproteomic coverage through sequential digestion by complementary proteases

    Journal: Analytical and bioanalytical chemistry

    doi: 10.1007/s00216-011-5466-5

    Phosphorylation sites identified by each approach. (a) Total number of distinct phosphorylation sites by method. Each experimental approach identified a larger number of phosphorylation sites than Glu-C only workflows. (b) Comparison of unique phosphorylation
    Figure Legend Snippet: Phosphorylation sites identified by each approach. (a) Total number of distinct phosphorylation sites by method. Each experimental approach identified a larger number of phosphorylation sites than Glu-C only workflows. (b) Comparison of unique phosphorylation

    Techniques Used:

    Experimental design and analysis of sequential digestion workflows. (a) Schematic diagram of the three sequential digestion approaches. GSPT closely resembles the traditional workflow with substitution of Glu-C for initial digestion and trypsin digestion
    Figure Legend Snippet: Experimental design and analysis of sequential digestion workflows. (a) Schematic diagram of the three sequential digestion approaches. GSPT closely resembles the traditional workflow with substitution of Glu-C for initial digestion and trypsin digestion

    Techniques Used:

    Evaluation of a Glu-C only based phosphoproteomic workflow. (a) In the traditional phosphoproteomic workflow, proteins are obtained from cell lysates and proteolytically digested with trypsin. The resultant peptides are separated into fractions via strong
    Figure Legend Snippet: Evaluation of a Glu-C only based phosphoproteomic workflow. (a) In the traditional phosphoproteomic workflow, proteins are obtained from cell lysates and proteolytically digested with trypsin. The resultant peptides are separated into fractions via strong

    Techniques Used:

    6) 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

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

    Techniques Used: Incubation

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

    Techniques Used: Incubation

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

    Techniques Used:

    Related Articles

    Modification:

    Article Title: Increasing phosphoproteomic coverage through sequential digestion by complementary proteases
    Article Snippet: .. Modified trypsin was from Promega (Madison, WI); Glu-C protease was from Worthington Biochemicals (Lakewood, NJ). .. Urea, Tris-HCl, CaCl2 , ammonium bicarbonate (NH4 HCO3 ), sodium fluoride (NaCl), potassium fluoride (KCl), potassium phosphate (KH2 PO4 ), phosphoric acid, sodium ortho-vanadate, sodium molybdate, sodium tartrate, beta-glycerophosphate, DL-dithiothreitol, iodoacetamide were from Sigma-Aldrich (St. Louis, MO).

    Immunoprecipitation:

    Article Title: Identification of a region in the coiled-coil domain of Smc3 that is essential for cohesin activity
    Article Snippet: .. Limited proteolysis Immunoprecipitated Smc3-3V5, either wild-type or L217P, were incubated in 20 μl of buffer (50 mM Tris–HCl (pH 8.0) and 1 mM MgCl2 ) with 5 mg/ml V8 protease (Worthington Biochemical), with or without 1 mM ATP (Sigma). ..

    Incubation:

    Article Title: Identification of a region in the coiled-coil domain of Smc3 that is essential for cohesin activity
    Article Snippet: .. Smc3-3V5 or smc3-L217P-3V5 were precipitated from asynchronous cell cultures and incubated with V8 protease, with or without ATP. .. The reaction products were analyzed by western blot with antibodies against V5.

    Article Title: Identification of a region in the coiled-coil domain of Smc3 that is essential for cohesin activity
    Article Snippet: .. Limited proteolysis Immunoprecipitated Smc3-3V5, either wild-type or L217P, were incubated in 20 μl of buffer (50 mM Tris–HCl (pH 8.0) and 1 mM MgCl2 ) with 5 mg/ml V8 protease (Worthington Biochemical), with or without 1 mM ATP (Sigma). ..

    other:

    Article Title: Increasing phosphoproteomic coverage through sequential digestion by complementary proteases
    Article Snippet: Briefly, a single, large pool of NCI-H23 cells were lysed in 8M urea, and their proteins were digested using Glu-C protease, desalted, and lyophilized in three equal (5mg) aliquots.

    Activity Assay:

    Article Title: Structural and Functional Characterization of Mature Forms of Metalloprotease E495 from Arctic Sea-Ice Bacterium Pseudoalteromonas sp. SM495
    Article Snippet: .. The protease activity to gelatin was determined with the method provided by Worthington Biochemical Co. . .. The degradation ability of E495-M and E495-M-C1 to CPC, APC and gamma globulin were analyzed using SDS-PAGE.

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