mnase  (New England Biolabs)


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    Structured Review

    New England Biolabs mnase
    Dynamics of H2B K34ub nucleosomes. ( A ) Nucleosomes assembled on 147 bp 601 <t>DNA</t> were resolved in native PAGE under ionic and temperature conditions indicated on top. For the middle panel, although electrophoresis was performed at ∼26°C, the temperature at the glass surface was ∼35–37°C due to higher conductivity of the buffer. All gels were pre-electrophoresed in the relevant buffer. ( B ) Unmodified and H2B K34ub nucleosomes /hexasomes assembled on 177 bp 601 were digested with <t>MNase</t> at 26°C or 37°C and DNA was resolved in 6.5% PAGE and stained with SYBR Gold.
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    Images

    1) Product Images from "Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky526

    Dynamics of H2B K34ub nucleosomes. ( A ) Nucleosomes assembled on 147 bp 601 DNA were resolved in native PAGE under ionic and temperature conditions indicated on top. For the middle panel, although electrophoresis was performed at ∼26°C, the temperature at the glass surface was ∼35–37°C due to higher conductivity of the buffer. All gels were pre-electrophoresed in the relevant buffer. ( B ) Unmodified and H2B K34ub nucleosomes /hexasomes assembled on 177 bp 601 were digested with MNase at 26°C or 37°C and DNA was resolved in 6.5% PAGE and stained with SYBR Gold.
    Figure Legend Snippet: Dynamics of H2B K34ub nucleosomes. ( A ) Nucleosomes assembled on 147 bp 601 DNA were resolved in native PAGE under ionic and temperature conditions indicated on top. For the middle panel, although electrophoresis was performed at ∼26°C, the temperature at the glass surface was ∼35–37°C due to higher conductivity of the buffer. All gels were pre-electrophoresed in the relevant buffer. ( B ) Unmodified and H2B K34ub nucleosomes /hexasomes assembled on 177 bp 601 were digested with MNase at 26°C or 37°C and DNA was resolved in 6.5% PAGE and stained with SYBR Gold.

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

    2) Product Images from "Reconstitution of eukaryotic chromosomes and manipulation of DNA N6-methyladenine alters chromatin and gene expression"

    Article Title: Reconstitution of eukaryotic chromosomes and manipulation of DNA N6-methyladenine alters chromatin and gene expression

    Journal: bioRxiv

    doi: 10.1101/475384

    Effects of 6mA on nucleosome organization in vitro and in vivo Experimental workflow for the generation of mini-genome DNA from native genomic DNA, and subsequent analysis by MNase-seq. Agarose gel analysis of Oxytricha gDNA (‘Native’) and mini-genome DNA before chromatin assembly. Methylated regions in the genome exhibit lower nucleosome occupancy in vitro but not in vivo . MNase-seq (nucleosome occupancy) and 6mA IP-seq coverage were calculated within overlapping 51bp windows across the 98 assayed chromosomes. Windows were binned according to the number of 6mA residues within. The in vitro MNase-seq coverage from chromatinized native gDNA (“+” 6mA) was divided by the corresponding coverage from chromatinized mini-genome DNA (“-“ 6mA) to obtain the fold change in nucleosome occupancy in each window (“+” histones). A subtraction was performed on these datasets to obtain the difference in nucleosome occupancy in vitro . Identical DNA sequences were compared for each calculation. Naked native gDNA and mini-genome DNA were also MNase-digested, sequenced and analyzed in the same manner, to control for MNase sequence preferences (“-“ histones). Nucleosome occupancy in vivo corresponds to MNase-seq coverage from wild type and mta1 mutant cells. P -values were calculated using a two-sample unequal variance t-test. N.S denotes “non-significant”, with p > 0.05. Tracks of 6mA distribution and MNase-seq coverage reveal a reduction in nucleosome occupancy at methylated loci in vitro (black arrowheads). 6mA “+” strand and “-“ strand tracks refer to SMRT-seq base calls from wild type genomic DNA. For the in vitro MNase-seq tracks, “+ 6mA” refers to chromatin assembled on Oxytricha gDNA, while “-6mA” denotes chromatin assembled on mini-genome DNA. Similar trends are observed for both Oxytricha and Xenopus histones. Vertical axis for SMRT-seq data denotes confidence score [-10 log(p-value)] of detection of the methylation event, while that for in vitro MNase-seq data denotes normalized read coverage. Tracks of 6mA distribution and MNase-seq coverage in vivo reveal no change in nucleosome occupancy in linker regions despite loss of 6mA in mta1 mutants. Vertical axis for SMRT-seq tracks denote confidence score [-10 log(p-value)] of detection of the methylation event, while that for in vivo MNase-seq tracks denote normalized read coverage.
    Figure Legend Snippet: Effects of 6mA on nucleosome organization in vitro and in vivo Experimental workflow for the generation of mini-genome DNA from native genomic DNA, and subsequent analysis by MNase-seq. Agarose gel analysis of Oxytricha gDNA (‘Native’) and mini-genome DNA before chromatin assembly. Methylated regions in the genome exhibit lower nucleosome occupancy in vitro but not in vivo . MNase-seq (nucleosome occupancy) and 6mA IP-seq coverage were calculated within overlapping 51bp windows across the 98 assayed chromosomes. Windows were binned according to the number of 6mA residues within. The in vitro MNase-seq coverage from chromatinized native gDNA (“+” 6mA) was divided by the corresponding coverage from chromatinized mini-genome DNA (“-“ 6mA) to obtain the fold change in nucleosome occupancy in each window (“+” histones). A subtraction was performed on these datasets to obtain the difference in nucleosome occupancy in vitro . Identical DNA sequences were compared for each calculation. Naked native gDNA and mini-genome DNA were also MNase-digested, sequenced and analyzed in the same manner, to control for MNase sequence preferences (“-“ histones). Nucleosome occupancy in vivo corresponds to MNase-seq coverage from wild type and mta1 mutant cells. P -values were calculated using a two-sample unequal variance t-test. N.S denotes “non-significant”, with p > 0.05. Tracks of 6mA distribution and MNase-seq coverage reveal a reduction in nucleosome occupancy at methylated loci in vitro (black arrowheads). 6mA “+” strand and “-“ strand tracks refer to SMRT-seq base calls from wild type genomic DNA. For the in vitro MNase-seq tracks, “+ 6mA” refers to chromatin assembled on Oxytricha gDNA, while “-6mA” denotes chromatin assembled on mini-genome DNA. Similar trends are observed for both Oxytricha and Xenopus histones. Vertical axis for SMRT-seq data denotes confidence score [-10 log(p-value)] of detection of the methylation event, while that for in vitro MNase-seq data denotes normalized read coverage. Tracks of 6mA distribution and MNase-seq coverage in vivo reveal no change in nucleosome occupancy in linker regions despite loss of 6mA in mta1 mutants. Vertical axis for SMRT-seq tracks denote confidence score [-10 log(p-value)] of detection of the methylation event, while that for in vivo MNase-seq tracks denote normalized read coverage.

    Techniques Used: In Vitro, In Vivo, Agarose Gel Electrophoresis, Methylation, Sequencing, Mutagenesis

    Epigenomic profiles of chromatin, transcription and DNA methylation in Oxytricha chromosomes Meta-chromosome plots overlaying in vivo MNase-seq (nucleosome occupancy), SMRT-seq (6mA), and 5’-complete cDNA sequencing data (transcription start sites; TSSs) at Oxytricha macronuclear chromosome ends. Heterodimeric telomere end- binding protein complexes protect each end in vivo and are denoted as orange ovals. Horizontal red bar denotes the promoter. The 5’ chromosome end is designated as being proximal to TSSs. +1, +2, and +3 nucleosomes are labeled relative to the 5’ chromosome end. Vertical axis: “Nucleosome occupancy” denotes MNase-seq read coverage; “6mA” denotes total number of detected 6mA marks; “Transcription start sites” denotes total number of called TSSs. Histograms of the total number of 6mA marks within each linker in Oxytricha chromosomes. Distinct linkers are depicted as horizontal bold blue lines. Transcriptional activity is positively correlated with 6mA levels. RNAseq data are derived from poly(A)-enriched RNA. Genes are sorted into 8 groups, according to the total number of 6mA marks between 0 bp to 800 bp downstream of the TSS. FPKM = Fragments per Kilobase of transcript per Million mapped RNAseq reads. Notch in the boxplot denotes median, ends of boxplot denote first and third quartiles, upper whisker denotes third quantile + 1.5 × interquartile range, and lower whisker denotes data quartile 1 − 1.5 × interquartile range. Composite analysis of 65,107 methylation sites reveals that 6mA (marked with * ) occurs within an 5’-ApT-3’ dinucleotide motif. Distribution of various 6mA dinucleotide motifs across the genome. Asterisk indicates DNA N6-methyladenine.
    Figure Legend Snippet: Epigenomic profiles of chromatin, transcription and DNA methylation in Oxytricha chromosomes Meta-chromosome plots overlaying in vivo MNase-seq (nucleosome occupancy), SMRT-seq (6mA), and 5’-complete cDNA sequencing data (transcription start sites; TSSs) at Oxytricha macronuclear chromosome ends. Heterodimeric telomere end- binding protein complexes protect each end in vivo and are denoted as orange ovals. Horizontal red bar denotes the promoter. The 5’ chromosome end is designated as being proximal to TSSs. +1, +2, and +3 nucleosomes are labeled relative to the 5’ chromosome end. Vertical axis: “Nucleosome occupancy” denotes MNase-seq read coverage; “6mA” denotes total number of detected 6mA marks; “Transcription start sites” denotes total number of called TSSs. Histograms of the total number of 6mA marks within each linker in Oxytricha chromosomes. Distinct linkers are depicted as horizontal bold blue lines. Transcriptional activity is positively correlated with 6mA levels. RNAseq data are derived from poly(A)-enriched RNA. Genes are sorted into 8 groups, according to the total number of 6mA marks between 0 bp to 800 bp downstream of the TSS. FPKM = Fragments per Kilobase of transcript per Million mapped RNAseq reads. Notch in the boxplot denotes median, ends of boxplot denote first and third quartiles, upper whisker denotes third quantile + 1.5 × interquartile range, and lower whisker denotes data quartile 1 − 1.5 × interquartile range. Composite analysis of 65,107 methylation sites reveals that 6mA (marked with * ) occurs within an 5’-ApT-3’ dinucleotide motif. Distribution of various 6mA dinucleotide motifs across the genome. Asterisk indicates DNA N6-methyladenine.

    Techniques Used: DNA Methylation Assay, In Vivo, Sequencing, Binding Assay, Labeling, Activity Assay, Derivative Assay, Whisker Assay, Methylation

    Quantitative modulation of nucleosome occupancy by 6mA in synthetic chromosomes Experimental workflow. Chromatin is assembled using either salt dialysis or the NAP1 histone chaperone. Italicized blue steps are selectively included. Tiling qPCR analysis of nucleosome occupancy in the synthetic chromosome Contig1781.0, with cognate 6mA sites. Horizontal grey box represents the annotated gene within Contig1781.0, with vertical grey lines depicting 6mA positions as obtained from Figure 5A. Horizontal blue bars span ~100bp regions amplified by qPCR primer pairs. Red horizontal lines and vertical bars represent the region containing 6mA. ‘Hemi methyl’ chromosomes contain 6mA on the antisense and sense strands, respectively, while the ‘Full methyl’ chromosome has 6mA on both strands. Nucleosome occupancy represents normalized qPCR signal at each locus (see Methods). For each qPCR locus, “difference” = nucleosome occupancy in methylated chromosome – nucleosome occupancy in “no methyl chromosome”; “fold change” = nucleosome occupancy in methylated chromosome / nucleosome occupancy in no methyl chromosome. Black arrowheads denote the decrease in nucleosome occupancy specifically at the 6mA cluster. Tiling qPCR analysis of nucleosome occupancy in synthetic chromosome Contig1781.0 with ectopic 6mA sites. Vertical black lines illustrate possible 6mA sites installed enzymatically. Red arrowheads denote the decrease in nucleosome occupancy in the ectopically methylated region, while black arrowheads denote the position of cognate 6mA sites (not in this construct). The chromatin remodeler ACF can partially overcome the effects of 6mA on nucleosome organization in an ATP-dependent manner. Chromatin is assembled by salt dialysis on synthetic chromosomes from panel B, and subsequently incubated with ACF and/or ATP. ACF equalizes nucleosome occupancy between the 6mA cluster and flanking regions in the presence of ATP (black line), resulting in a relative increase in nucleosome occupancy across the methylated region. Nucleosome occupancy at the methylated region is not restored to the same level as the unmethylated control in the presence of ACF and ATP (black arrowheads). (E) Chromatin is assembled on native gDNA (“+” 6mA) and mini-genome DNA (“-“ 6mA) using NAP1 in the presence of ACF and/or ATP, and subsequently analyzed using MNase-seq. Sliding window fold change in nucleosome occupancy between native gDNA and mini-genome DNA is calculated as in Figure 4C. ACF acts in an ATP-dependent manner to restore nucleosome occupancy in methylated DNA windows. P -values were calculated using a two-sample unequal variance t-test.
    Figure Legend Snippet: Quantitative modulation of nucleosome occupancy by 6mA in synthetic chromosomes Experimental workflow. Chromatin is assembled using either salt dialysis or the NAP1 histone chaperone. Italicized blue steps are selectively included. Tiling qPCR analysis of nucleosome occupancy in the synthetic chromosome Contig1781.0, with cognate 6mA sites. Horizontal grey box represents the annotated gene within Contig1781.0, with vertical grey lines depicting 6mA positions as obtained from Figure 5A. Horizontal blue bars span ~100bp regions amplified by qPCR primer pairs. Red horizontal lines and vertical bars represent the region containing 6mA. ‘Hemi methyl’ chromosomes contain 6mA on the antisense and sense strands, respectively, while the ‘Full methyl’ chromosome has 6mA on both strands. Nucleosome occupancy represents normalized qPCR signal at each locus (see Methods). For each qPCR locus, “difference” = nucleosome occupancy in methylated chromosome – nucleosome occupancy in “no methyl chromosome”; “fold change” = nucleosome occupancy in methylated chromosome / nucleosome occupancy in no methyl chromosome. Black arrowheads denote the decrease in nucleosome occupancy specifically at the 6mA cluster. Tiling qPCR analysis of nucleosome occupancy in synthetic chromosome Contig1781.0 with ectopic 6mA sites. Vertical black lines illustrate possible 6mA sites installed enzymatically. Red arrowheads denote the decrease in nucleosome occupancy in the ectopically methylated region, while black arrowheads denote the position of cognate 6mA sites (not in this construct). The chromatin remodeler ACF can partially overcome the effects of 6mA on nucleosome organization in an ATP-dependent manner. Chromatin is assembled by salt dialysis on synthetic chromosomes from panel B, and subsequently incubated with ACF and/or ATP. ACF equalizes nucleosome occupancy between the 6mA cluster and flanking regions in the presence of ATP (black line), resulting in a relative increase in nucleosome occupancy across the methylated region. Nucleosome occupancy at the methylated region is not restored to the same level as the unmethylated control in the presence of ACF and ATP (black arrowheads). (E) Chromatin is assembled on native gDNA (“+” 6mA) and mini-genome DNA (“-“ 6mA) using NAP1 in the presence of ACF and/or ATP, and subsequently analyzed using MNase-seq. Sliding window fold change in nucleosome occupancy between native gDNA and mini-genome DNA is calculated as in Figure 4C. ACF acts in an ATP-dependent manner to restore nucleosome occupancy in methylated DNA windows. P -values were calculated using a two-sample unequal variance t-test.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Methylation, Construct, Incubation

    3) Product Images from "Regulatory architecture of the RCA gene cluster captures an intragenic TAD boundary and enhancer elements in B cells"

    Article Title: Regulatory architecture of the RCA gene cluster captures an intragenic TAD boundary and enhancer elements in B cells

    Journal: bioRxiv

    doi: 10.1101/2020.02.16.941070

    BEN-2 shows B cell-specific nucleosome occupancy, chromatin accessibility and enrichment for the H3K27ac active enhancer histone mark across a panel of B cell lines. A. Nucleosome occupancy at BEN-2 as measured by ChART-PCR with MNase digestion. Data was normalised to the inaccessible SFTPA2 gene promoter such that a value of 1.0 represents fully compacted nucleosomes, and lower values indicate less compacted nucleosomes. B. Chromatin accessibility at BEN-2 as measured by ChART-PCR with DNase I digestion. Data have been normalised to the inaccessible SFTPA2 gene promoter. C. H3K27ac enrichment at BEN-2 as determined by ChIP-qPCR using the percent input method. Grey bars indicate H3K27ac enrichment at the target locus, and black bars show enrichment using a non-specific IgG control antibody. All data are presented as mean ± SEM from at least 3 biological replicates.
    Figure Legend Snippet: BEN-2 shows B cell-specific nucleosome occupancy, chromatin accessibility and enrichment for the H3K27ac active enhancer histone mark across a panel of B cell lines. A. Nucleosome occupancy at BEN-2 as measured by ChART-PCR with MNase digestion. Data was normalised to the inaccessible SFTPA2 gene promoter such that a value of 1.0 represents fully compacted nucleosomes, and lower values indicate less compacted nucleosomes. B. Chromatin accessibility at BEN-2 as measured by ChART-PCR with DNase I digestion. Data have been normalised to the inaccessible SFTPA2 gene promoter. C. H3K27ac enrichment at BEN-2 as determined by ChIP-qPCR using the percent input method. Grey bars indicate H3K27ac enrichment at the target locus, and black bars show enrichment using a non-specific IgG control antibody. All data are presented as mean ± SEM from at least 3 biological replicates.

    Techniques Used: Polymerase Chain Reaction, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    4) Product Images from "Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans"

    Article Title: Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-11-465

    DAM accessibility correlates with nucleosome positions . This figure shows the superimposition of average DAM accessibility versus average MNase accessibility around the transcription start site (TSS) of 3,904 strongly expressed C. elegans genes. The dotted red curve represents a moving average of the muscle profile with a sliding window of 400 nucleotides. Positioned H3K4me2/3 nucleosomes are represented by ovals above the picture of the generic gene. Numbers on the nucleosomes indicate their positions relative to the TSS. [NDR = nucleosome depleted region]
    Figure Legend Snippet: DAM accessibility correlates with nucleosome positions . This figure shows the superimposition of average DAM accessibility versus average MNase accessibility around the transcription start site (TSS) of 3,904 strongly expressed C. elegans genes. The dotted red curve represents a moving average of the muscle profile with a sliding window of 400 nucleotides. Positioned H3K4me2/3 nucleosomes are represented by ovals above the picture of the generic gene. Numbers on the nucleosomes indicate their positions relative to the TSS. [NDR = nucleosome depleted region]

    Techniques Used:

    5) Product Images from "Nucleosomes Are Stably Evicted from Enhancers but Not Promoters upon Induction of Certain Pro-Inflammatory Genes in Mouse Macrophages"

    Article Title: Nucleosomes Are Stably Evicted from Enhancers but Not Promoters upon Induction of Certain Pro-Inflammatory Genes in Mouse Macrophages

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0093971

    PolII and TBP binding in the fraction of IL12B and IL1A promoters in a population of induced BMDMs that is nucleosome-free. (A and B), ChIP experiments were performed as described in the legend of Figure 2F with antibodies that detect (A) PolII or (B) TBP in BMDMs before (dark blue bars), and upon 1.5 h (yellow) or 3 h (red) LPS induction. Cross-linked chromatin was either untreated (solid bars), or lightly digested with MNase (hatched bars) as described in the Materials and Methods . The data was normalized to a region in the KIT promoter and genomic locations are indicated. The experiment was performed twice and error bars indicating the SEM are shown.
    Figure Legend Snippet: PolII and TBP binding in the fraction of IL12B and IL1A promoters in a population of induced BMDMs that is nucleosome-free. (A and B), ChIP experiments were performed as described in the legend of Figure 2F with antibodies that detect (A) PolII or (B) TBP in BMDMs before (dark blue bars), and upon 1.5 h (yellow) or 3 h (red) LPS induction. Cross-linked chromatin was either untreated (solid bars), or lightly digested with MNase (hatched bars) as described in the Materials and Methods . The data was normalized to a region in the KIT promoter and genomic locations are indicated. The experiment was performed twice and error bars indicating the SEM are shown.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation

    6) Product Images from "HITS-CLIP Analysis Uncovers a Link between the Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein and Host Pre-mRNA Metabolism"

    Article Title: HITS-CLIP Analysis Uncovers a Link between the Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein and Host Pre-mRNA Metabolism

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004652

    Isolation of cross-linked ORF57-RNA complexes for HITS-CLIP analysis. Top Nitrocellulose membrane from an ORF57 immunoprecipitation performed under HITS-CLIP conditions. Cross-linked RNA fragments were end-labeled with 32 P to be visualized by Phosphoimager. Cells were induced to undergo lytic reactivation, exposed to UV and/or treated with high or low concentrations of MNase as indicated. The samples lacking an ORF57 antibody (lane 1) were precipitated with pre-bleed antibodies from the same rabbit. Lanes 6 and 7 are a dark exposure of lanes 4 and 5. The dashed white box indicates the position of the ORF57 complex cut from the membrane for library preparation. Bottom Western blot of the same HITS-CLIP samples shown in the top panel. Affinity purified rabbit anti-ORF57 was used to detect ORF57. Positions of molecular weight markers are shown on the left. On the right, a single asterisk marks the position of a contaminating ~37 kDa protein; double and triple asterisks mark positions of putative ORF57 homodimers and homotrimers bound to the same RNA. The doublet is likely due to an ORF57 cleavage product [ 104 ].
    Figure Legend Snippet: Isolation of cross-linked ORF57-RNA complexes for HITS-CLIP analysis. Top Nitrocellulose membrane from an ORF57 immunoprecipitation performed under HITS-CLIP conditions. Cross-linked RNA fragments were end-labeled with 32 P to be visualized by Phosphoimager. Cells were induced to undergo lytic reactivation, exposed to UV and/or treated with high or low concentrations of MNase as indicated. The samples lacking an ORF57 antibody (lane 1) were precipitated with pre-bleed antibodies from the same rabbit. Lanes 6 and 7 are a dark exposure of lanes 4 and 5. The dashed white box indicates the position of the ORF57 complex cut from the membrane for library preparation. Bottom Western blot of the same HITS-CLIP samples shown in the top panel. Affinity purified rabbit anti-ORF57 was used to detect ORF57. Positions of molecular weight markers are shown on the left. On the right, a single asterisk marks the position of a contaminating ~37 kDa protein; double and triple asterisks mark positions of putative ORF57 homodimers and homotrimers bound to the same RNA. The doublet is likely due to an ORF57 cleavage product [ 104 ].

    Techniques Used: Isolation, Cross-linking Immunoprecipitation, Immunoprecipitation, Labeling, Western Blot, Affinity Purification, Molecular Weight

    7) Product Images from "Organization of DNA in a bacterial nucleoid"

    Article Title: Organization of DNA in a bacterial nucleoid

    Journal: BMC Microbiology

    doi: 10.1186/s12866-016-0637-3

    A possible model of low-level nucleoid organization. a A fragment of EM image of E. coli nucleoid (adapted from [ 21 ]). b Genomic DNA (gray) forms loops with A-tract clusters (cyan) located at apexes and MNase resistant fragments (black) occupying loop ‘stems’. c A blow-up of a DNA loop, containing several A-tracts (cyan) and two MNase resistant fragments (black)
    Figure Legend Snippet: A possible model of low-level nucleoid organization. a A fragment of EM image of E. coli nucleoid (adapted from [ 21 ]). b Genomic DNA (gray) forms loops with A-tract clusters (cyan) located at apexes and MNase resistant fragments (black) occupying loop ‘stems’. c A blow-up of a DNA loop, containing several A-tracts (cyan) and two MNase resistant fragments (black)

    Techniques Used:

    Analysis of the DNA fragments from in vivo MNase digestion of nucleoid in wild type E. coli ( a ) and in vitro MNase digestion of purified genomic DNA ( b ) in agarose gel. a Wild type cells with the empty vector (lanes 2, 3) and MNase-expressing vector (lanes 4-8) were supplemented with arabinose (lane 6), CaCl 2 (lane 5) or both (lanes 3, 7, 8). Digestion reactions were stopped 1 minute (lane 7) or 5 minutes (lanes 3, 5, 8) after CaCl 2 was added. Lanes 1 and 9 show DNA molecular weight marker. b Lane 1, purified wild type genomic DNA; lanes 2-6, a time course of in vitro MNase digestion of the wild type genomic DNA; lane 7, DNA molecular marker
    Figure Legend Snippet: Analysis of the DNA fragments from in vivo MNase digestion of nucleoid in wild type E. coli ( a ) and in vitro MNase digestion of purified genomic DNA ( b ) in agarose gel. a Wild type cells with the empty vector (lanes 2, 3) and MNase-expressing vector (lanes 4-8) were supplemented with arabinose (lane 6), CaCl 2 (lane 5) or both (lanes 3, 7, 8). Digestion reactions were stopped 1 minute (lane 7) or 5 minutes (lanes 3, 5, 8) after CaCl 2 was added. Lanes 1 and 9 show DNA molecular weight marker. b Lane 1, purified wild type genomic DNA; lanes 2-6, a time course of in vitro MNase digestion of the wild type genomic DNA; lane 7, DNA molecular marker

    Techniques Used: In Vivo, In Vitro, Purification, Agarose Gel Electrophoresis, Plasmid Preparation, Expressing, Molecular Weight, Marker

    Genome-wide distribution of sequenced tags. a Tag frequencies for the entire E. coli genome. Frequencies of tags mapped on the positive and negative strands are shown with red and blue bars respectively. a , c Schematic illustrations of tag cross-correlation ( b ) and auto-correlation analyses ( c ). MNase resistant fragments are shown with grey rectangles. Vertical red and blue arrows represent 5’-ends of the digestion fragments mapping to the DNA positive and negative strands respectively
    Figure Legend Snippet: Genome-wide distribution of sequenced tags. a Tag frequencies for the entire E. coli genome. Frequencies of tags mapped on the positive and negative strands are shown with red and blue bars respectively. a , c Schematic illustrations of tag cross-correlation ( b ) and auto-correlation analyses ( c ). MNase resistant fragments are shown with grey rectangles. Vertical red and blue arrows represent 5’-ends of the digestion fragments mapping to the DNA positive and negative strands respectively

    Techniques Used: Genome Wide

    8) Product Images from "An Improved Strategy to Recover Large Fragments of Functional Human Neutrophil Extracellular Traps"

    Article Title: An Improved Strategy to Recover Large Fragments of Functional Human Neutrophil Extracellular Traps

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2013.00166

    DNA nucleases induce NET digestion . (A) Migration profile of pure λDNA after digestion with 4 U/mL DNase, MNase, or Alu-I. (B) Alu-I, DNase, and MNase dose-effects on NET dsDNA obtained after A23187 stimulation of PMN. Incubation with the restriction enzymes lasted 20 min at 37°C. DNA migration took place in 0.8% agarose gel containing ethidium bromide.
    Figure Legend Snippet: DNA nucleases induce NET digestion . (A) Migration profile of pure λDNA after digestion with 4 U/mL DNase, MNase, or Alu-I. (B) Alu-I, DNase, and MNase dose-effects on NET dsDNA obtained after A23187 stimulation of PMN. Incubation with the restriction enzymes lasted 20 min at 37°C. DNA migration took place in 0.8% agarose gel containing ethidium bromide.

    Techniques Used: Migration, Incubation, Agarose Gel Electrophoresis

    9) Product Images from "Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum"

    Article Title: Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum

    Journal: EMBO Reports

    doi: 10.15252/embr.201846331

    Ectopically expressed histone H3p localizes to the nucleus during parasite asexual development and incorporates into nucleosomes Indirect immunofluorescence assays were performed to determine the localization of ectopically expressed PfH3p‐HA in ring (R), trophozoite (T), and schizont (S) stages of Plasmodium falciparum asexual growth. PfH3p‐HA was detected using anti‐HA antibodies (green) and endogenous histone H3 with anti‐histone H3 N‐terminal antibodies (red). DAPI (blue) was used to stain the nucleus. Scale bar = 5 μm. Nuclei isolated from wild‐type (WT) or PfH3p‐HA‐expressing (WT + PfH3p‐HA) schizont‐stage parasites were treated with 4 U/ml of micrococcal nuclease (MNase) for the indicated amounts of time, the DNA purified and migrated on a 2% agarose gel, and stained with ethidium bromide. Mononucleosomes purified after 10 min of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis and either stained with Coomassie Brilliant Blue (C.B.) or visualized by immunoblotting with anti‐HA (α‐HA) or anti‐C‐terminal histone H3 (α‐H3c) antibodies. Co‐immunoprecipitation (IP) experiments of purified mononucleosomes obtained from wild‐type (WT) or transfected (WT + PfH3p‐HA) schizont‐stage parasites were performed with either anti‐HA antibodies or mouse IgG. Immunoprecipitated products (right panel) were analyzed by immunoblotting using anti‐HA or anti‐histone H4 antibodies. Source data are available online for this figure.
    Figure Legend Snippet: Ectopically expressed histone H3p localizes to the nucleus during parasite asexual development and incorporates into nucleosomes Indirect immunofluorescence assays were performed to determine the localization of ectopically expressed PfH3p‐HA in ring (R), trophozoite (T), and schizont (S) stages of Plasmodium falciparum asexual growth. PfH3p‐HA was detected using anti‐HA antibodies (green) and endogenous histone H3 with anti‐histone H3 N‐terminal antibodies (red). DAPI (blue) was used to stain the nucleus. Scale bar = 5 μm. Nuclei isolated from wild‐type (WT) or PfH3p‐HA‐expressing (WT + PfH3p‐HA) schizont‐stage parasites were treated with 4 U/ml of micrococcal nuclease (MNase) for the indicated amounts of time, the DNA purified and migrated on a 2% agarose gel, and stained with ethidium bromide. Mononucleosomes purified after 10 min of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis and either stained with Coomassie Brilliant Blue (C.B.) or visualized by immunoblotting with anti‐HA (α‐HA) or anti‐C‐terminal histone H3 (α‐H3c) antibodies. Co‐immunoprecipitation (IP) experiments of purified mononucleosomes obtained from wild‐type (WT) or transfected (WT + PfH3p‐HA) schizont‐stage parasites were performed with either anti‐HA antibodies or mouse IgG. Immunoprecipitated products (right panel) were analyzed by immunoblotting using anti‐HA or anti‐histone H4 antibodies. Source data are available online for this figure.

    Techniques Used: Immunofluorescence, Staining, Isolation, Expressing, Purification, Agarose Gel Electrophoresis, Polyacrylamide Gel Electrophoresis, Immunoprecipitation, Transfection

    10) Product Images from "Reconstitution of eukaryotic chromosomes and manipulation of DNA N6-methyladenine alters chromatin and gene expression"

    Article Title: Reconstitution of eukaryotic chromosomes and manipulation of DNA N6-methyladenine alters chromatin and gene expression

    Journal: bioRxiv

    doi: 10.1101/475384

    Effects of 6mA on nucleosome organization in vitro and in vivo Experimental workflow for the generation of mini-genome DNA from native genomic DNA, and subsequent analysis by MNase-seq. Agarose gel analysis of Oxytricha gDNA (‘Native’) and mini-genome DNA before chromatin assembly. Methylated regions in the genome exhibit lower nucleosome occupancy in vitro but not in vivo . MNase-seq (nucleosome occupancy) and 6mA IP-seq coverage were calculated within overlapping 51bp windows across the 98 assayed chromosomes. Windows were binned according to the number of 6mA residues within. The in vitro MNase-seq coverage from chromatinized native gDNA (“+” 6mA) was divided by the corresponding coverage from chromatinized mini-genome DNA (“-“ 6mA) to obtain the fold change in nucleosome occupancy in each window (“+” histones). A subtraction was performed on these datasets to obtain the difference in nucleosome occupancy in vitro . Identical DNA sequences were compared for each calculation. Naked native gDNA and mini-genome DNA were also MNase-digested, sequenced and analyzed in the same manner, to control for MNase sequence preferences (“-“ histones). Nucleosome occupancy in vivo corresponds to MNase-seq coverage from wild type and mta1 mutant cells. P -values were calculated using a two-sample unequal variance t-test. N.S denotes “non-significant”, with p > 0.05. Tracks of 6mA distribution and MNase-seq coverage reveal a reduction in nucleosome occupancy at methylated loci in vitro (black arrowheads). 6mA “+” strand and “-“ strand tracks refer to SMRT-seq base calls from wild type genomic DNA. For the in vitro MNase-seq tracks, “+ 6mA” refers to chromatin assembled on Oxytricha gDNA, while “-6mA” denotes chromatin assembled on mini-genome DNA. Similar trends are observed for both Oxytricha and Xenopus histones. Vertical axis for SMRT-seq data denotes confidence score [-10 log(p-value)] of detection of the methylation event, while that for in vitro MNase-seq data denotes normalized read coverage. Tracks of 6mA distribution and MNase-seq coverage in vivo reveal no change in nucleosome occupancy in linker regions despite loss of 6mA in mta1 mutants. Vertical axis for SMRT-seq tracks denote confidence score [-10 log(p-value)] of detection of the methylation event, while that for in vivo MNase-seq tracks denote normalized read coverage.
    Figure Legend Snippet: Effects of 6mA on nucleosome organization in vitro and in vivo Experimental workflow for the generation of mini-genome DNA from native genomic DNA, and subsequent analysis by MNase-seq. Agarose gel analysis of Oxytricha gDNA (‘Native’) and mini-genome DNA before chromatin assembly. Methylated regions in the genome exhibit lower nucleosome occupancy in vitro but not in vivo . MNase-seq (nucleosome occupancy) and 6mA IP-seq coverage were calculated within overlapping 51bp windows across the 98 assayed chromosomes. Windows were binned according to the number of 6mA residues within. The in vitro MNase-seq coverage from chromatinized native gDNA (“+” 6mA) was divided by the corresponding coverage from chromatinized mini-genome DNA (“-“ 6mA) to obtain the fold change in nucleosome occupancy in each window (“+” histones). A subtraction was performed on these datasets to obtain the difference in nucleosome occupancy in vitro . Identical DNA sequences were compared for each calculation. Naked native gDNA and mini-genome DNA were also MNase-digested, sequenced and analyzed in the same manner, to control for MNase sequence preferences (“-“ histones). Nucleosome occupancy in vivo corresponds to MNase-seq coverage from wild type and mta1 mutant cells. P -values were calculated using a two-sample unequal variance t-test. N.S denotes “non-significant”, with p > 0.05. Tracks of 6mA distribution and MNase-seq coverage reveal a reduction in nucleosome occupancy at methylated loci in vitro (black arrowheads). 6mA “+” strand and “-“ strand tracks refer to SMRT-seq base calls from wild type genomic DNA. For the in vitro MNase-seq tracks, “+ 6mA” refers to chromatin assembled on Oxytricha gDNA, while “-6mA” denotes chromatin assembled on mini-genome DNA. Similar trends are observed for both Oxytricha and Xenopus histones. Vertical axis for SMRT-seq data denotes confidence score [-10 log(p-value)] of detection of the methylation event, while that for in vitro MNase-seq data denotes normalized read coverage. Tracks of 6mA distribution and MNase-seq coverage in vivo reveal no change in nucleosome occupancy in linker regions despite loss of 6mA in mta1 mutants. Vertical axis for SMRT-seq tracks denote confidence score [-10 log(p-value)] of detection of the methylation event, while that for in vivo MNase-seq tracks denote normalized read coverage.

    Techniques Used: In Vitro, In Vivo, Agarose Gel Electrophoresis, Methylation, Sequencing, Mutagenesis

    Epigenomic profiles of chromatin, transcription and DNA methylation in Oxytricha chromosomes Meta-chromosome plots overlaying in vivo MNase-seq (nucleosome occupancy), SMRT-seq (6mA), and 5’-complete cDNA sequencing data (transcription start sites; TSSs) at Oxytricha macronuclear chromosome ends. Heterodimeric telomere end- binding protein complexes protect each end in vivo and are denoted as orange ovals. Horizontal red bar denotes the promoter. The 5’ chromosome end is designated as being proximal to TSSs. +1, +2, and +3 nucleosomes are labeled relative to the 5’ chromosome end. Vertical axis: “Nucleosome occupancy” denotes MNase-seq read coverage; “6mA” denotes total number of detected 6mA marks; “Transcription start sites” denotes total number of called TSSs. Histograms of the total number of 6mA marks within each linker in Oxytricha chromosomes. Distinct linkers are depicted as horizontal bold blue lines. Transcriptional activity is positively correlated with 6mA levels. RNAseq data are derived from poly(A)-enriched RNA. Genes are sorted into 8 groups, according to the total number of 6mA marks between 0 bp to 800 bp downstream of the TSS. FPKM = Fragments per Kilobase of transcript per Million mapped RNAseq reads. Notch in the boxplot denotes median, ends of boxplot denote first and third quartiles, upper whisker denotes third quantile + 1.5 × interquartile range, and lower whisker denotes data quartile 1 − 1.5 × interquartile range. Composite analysis of 65,107 methylation sites reveals that 6mA (marked with * ) occurs within an 5’-ApT-3’ dinucleotide motif. Distribution of various 6mA dinucleotide motifs across the genome. Asterisk indicates DNA N6-methyladenine.
    Figure Legend Snippet: Epigenomic profiles of chromatin, transcription and DNA methylation in Oxytricha chromosomes Meta-chromosome plots overlaying in vivo MNase-seq (nucleosome occupancy), SMRT-seq (6mA), and 5’-complete cDNA sequencing data (transcription start sites; TSSs) at Oxytricha macronuclear chromosome ends. Heterodimeric telomere end- binding protein complexes protect each end in vivo and are denoted as orange ovals. Horizontal red bar denotes the promoter. The 5’ chromosome end is designated as being proximal to TSSs. +1, +2, and +3 nucleosomes are labeled relative to the 5’ chromosome end. Vertical axis: “Nucleosome occupancy” denotes MNase-seq read coverage; “6mA” denotes total number of detected 6mA marks; “Transcription start sites” denotes total number of called TSSs. Histograms of the total number of 6mA marks within each linker in Oxytricha chromosomes. Distinct linkers are depicted as horizontal bold blue lines. Transcriptional activity is positively correlated with 6mA levels. RNAseq data are derived from poly(A)-enriched RNA. Genes are sorted into 8 groups, according to the total number of 6mA marks between 0 bp to 800 bp downstream of the TSS. FPKM = Fragments per Kilobase of transcript per Million mapped RNAseq reads. Notch in the boxplot denotes median, ends of boxplot denote first and third quartiles, upper whisker denotes third quantile + 1.5 × interquartile range, and lower whisker denotes data quartile 1 − 1.5 × interquartile range. Composite analysis of 65,107 methylation sites reveals that 6mA (marked with * ) occurs within an 5’-ApT-3’ dinucleotide motif. Distribution of various 6mA dinucleotide motifs across the genome. Asterisk indicates DNA N6-methyladenine.

    Techniques Used: DNA Methylation Assay, In Vivo, Sequencing, Binding Assay, Labeling, Activity Assay, Derivative Assay, Whisker Assay, Methylation

    Quantitative modulation of nucleosome occupancy by 6mA in synthetic chromosomes Experimental workflow. Chromatin is assembled using either salt dialysis or the NAP1 histone chaperone. Italicized blue steps are selectively included. Tiling qPCR analysis of nucleosome occupancy in the synthetic chromosome Contig1781.0, with cognate 6mA sites. Horizontal grey box represents the annotated gene within Contig1781.0, with vertical grey lines depicting 6mA positions as obtained from Figure 5A. Horizontal blue bars span ~100bp regions amplified by qPCR primer pairs. Red horizontal lines and vertical bars represent the region containing 6mA. ‘Hemi methyl’ chromosomes contain 6mA on the antisense and sense strands, respectively, while the ‘Full methyl’ chromosome has 6mA on both strands. Nucleosome occupancy represents normalized qPCR signal at each locus (see Methods). For each qPCR locus, “difference” = nucleosome occupancy in methylated chromosome – nucleosome occupancy in “no methyl chromosome”; “fold change” = nucleosome occupancy in methylated chromosome / nucleosome occupancy in no methyl chromosome. Black arrowheads denote the decrease in nucleosome occupancy specifically at the 6mA cluster. Tiling qPCR analysis of nucleosome occupancy in synthetic chromosome Contig1781.0 with ectopic 6mA sites. Vertical black lines illustrate possible 6mA sites installed enzymatically. Red arrowheads denote the decrease in nucleosome occupancy in the ectopically methylated region, while black arrowheads denote the position of cognate 6mA sites (not in this construct). The chromatin remodeler ACF can partially overcome the effects of 6mA on nucleosome organization in an ATP-dependent manner. Chromatin is assembled by salt dialysis on synthetic chromosomes from panel B, and subsequently incubated with ACF and/or ATP. ACF equalizes nucleosome occupancy between the 6mA cluster and flanking regions in the presence of ATP (black line), resulting in a relative increase in nucleosome occupancy across the methylated region. Nucleosome occupancy at the methylated region is not restored to the same level as the unmethylated control in the presence of ACF and ATP (black arrowheads). (E) Chromatin is assembled on native gDNA (“+” 6mA) and mini-genome DNA (“-“ 6mA) using NAP1 in the presence of ACF and/or ATP, and subsequently analyzed using MNase-seq. Sliding window fold change in nucleosome occupancy between native gDNA and mini-genome DNA is calculated as in Figure 4C. ACF acts in an ATP-dependent manner to restore nucleosome occupancy in methylated DNA windows. P -values were calculated using a two-sample unequal variance t-test.
    Figure Legend Snippet: Quantitative modulation of nucleosome occupancy by 6mA in synthetic chromosomes Experimental workflow. Chromatin is assembled using either salt dialysis or the NAP1 histone chaperone. Italicized blue steps are selectively included. Tiling qPCR analysis of nucleosome occupancy in the synthetic chromosome Contig1781.0, with cognate 6mA sites. Horizontal grey box represents the annotated gene within Contig1781.0, with vertical grey lines depicting 6mA positions as obtained from Figure 5A. Horizontal blue bars span ~100bp regions amplified by qPCR primer pairs. Red horizontal lines and vertical bars represent the region containing 6mA. ‘Hemi methyl’ chromosomes contain 6mA on the antisense and sense strands, respectively, while the ‘Full methyl’ chromosome has 6mA on both strands. Nucleosome occupancy represents normalized qPCR signal at each locus (see Methods). For each qPCR locus, “difference” = nucleosome occupancy in methylated chromosome – nucleosome occupancy in “no methyl chromosome”; “fold change” = nucleosome occupancy in methylated chromosome / nucleosome occupancy in no methyl chromosome. Black arrowheads denote the decrease in nucleosome occupancy specifically at the 6mA cluster. Tiling qPCR analysis of nucleosome occupancy in synthetic chromosome Contig1781.0 with ectopic 6mA sites. Vertical black lines illustrate possible 6mA sites installed enzymatically. Red arrowheads denote the decrease in nucleosome occupancy in the ectopically methylated region, while black arrowheads denote the position of cognate 6mA sites (not in this construct). The chromatin remodeler ACF can partially overcome the effects of 6mA on nucleosome organization in an ATP-dependent manner. Chromatin is assembled by salt dialysis on synthetic chromosomes from panel B, and subsequently incubated with ACF and/or ATP. ACF equalizes nucleosome occupancy between the 6mA cluster and flanking regions in the presence of ATP (black line), resulting in a relative increase in nucleosome occupancy across the methylated region. Nucleosome occupancy at the methylated region is not restored to the same level as the unmethylated control in the presence of ACF and ATP (black arrowheads). (E) Chromatin is assembled on native gDNA (“+” 6mA) and mini-genome DNA (“-“ 6mA) using NAP1 in the presence of ACF and/or ATP, and subsequently analyzed using MNase-seq. Sliding window fold change in nucleosome occupancy between native gDNA and mini-genome DNA is calculated as in Figure 4C. ACF acts in an ATP-dependent manner to restore nucleosome occupancy in methylated DNA windows. P -values were calculated using a two-sample unequal variance t-test.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Methylation, Construct, Incubation

    11) Product Images from "Single cell quantification of ribosome occupancy in early mouse development"

    Article Title: Single cell quantification of ribosome occupancy in early mouse development

    Journal: bioRxiv

    doi: 10.1101/2021.12.07.471408

    Characterization of Ribo-ITP method and validation of efficacy in ultra-low input ribosome profiling a, Representative gel images highlighting inputs (I), RNAs recovered by Ribo-ITP (R), and gel electrophoresis (G) are shown. Four RNAs of 17, 21, 25, and 29 nt used in the experiment were radioactively labeled at their 5’end. Percent yield was calculated for the 25 nt RNA. b, Representative gel image of a size selection experiment. 100 ng of MNase-digested RNA from K562 cells (D) was used as an input for Ribo-ITP after the addition of the two fluorescent marker oligonucleotides (I). In a typical experiment, we collect the sample flanked by the two fluorescent nucleotide markers (Fraction 2 here). Here, we also collected the RNAs that eluted before the arrival of the shorter fluorescent marker (Fraction 1) as well as the RNAs that were located behind the longer fluorescent marker (Fraction 3), which typically remain in the channel. The percent yield of RNAs larger than the longer fluorescent marker oligonucleotide ( > ∼36 nt) (blue) and RNAs flanked by the markers (orange), corresponding to the size range of RPFs, are plotted for each fraction. c, Schematic of the sequencing library preparation protocol. In a single-tube reaction, isolated RPFs are 3’ dephosphorylated and poly(A)-tailed. A template-switching reverse transcriptase (RT) creates templates that incorporate UMI-containing adapters. d, Pairwise correlation of gene-level ribosome occupancy measured in conventional ribosome profiling and Ribo-ITP from human K562 cells. The left plot highlights two replicates of conventional ribosome profiling experiments from ∼10M cells. The middle plot is from two replicates of Ribo-ITP with ∼100 cells. For the plot on the right, we used the mean number of counts per million reads for each gene. The Spearman correlation coefficients between the gene-level ribosome occupancies are indicated on the top left corner.
    Figure Legend Snippet: Characterization of Ribo-ITP method and validation of efficacy in ultra-low input ribosome profiling a, Representative gel images highlighting inputs (I), RNAs recovered by Ribo-ITP (R), and gel electrophoresis (G) are shown. Four RNAs of 17, 21, 25, and 29 nt used in the experiment were radioactively labeled at their 5’end. Percent yield was calculated for the 25 nt RNA. b, Representative gel image of a size selection experiment. 100 ng of MNase-digested RNA from K562 cells (D) was used as an input for Ribo-ITP after the addition of the two fluorescent marker oligonucleotides (I). In a typical experiment, we collect the sample flanked by the two fluorescent nucleotide markers (Fraction 2 here). Here, we also collected the RNAs that eluted before the arrival of the shorter fluorescent marker (Fraction 1) as well as the RNAs that were located behind the longer fluorescent marker (Fraction 3), which typically remain in the channel. The percent yield of RNAs larger than the longer fluorescent marker oligonucleotide ( > ∼36 nt) (blue) and RNAs flanked by the markers (orange), corresponding to the size range of RPFs, are plotted for each fraction. c, Schematic of the sequencing library preparation protocol. In a single-tube reaction, isolated RPFs are 3’ dephosphorylated and poly(A)-tailed. A template-switching reverse transcriptase (RT) creates templates that incorporate UMI-containing adapters. d, Pairwise correlation of gene-level ribosome occupancy measured in conventional ribosome profiling and Ribo-ITP from human K562 cells. The left plot highlights two replicates of conventional ribosome profiling experiments from ∼10M cells. The middle plot is from two replicates of Ribo-ITP with ∼100 cells. For the plot on the right, we used the mean number of counts per million reads for each gene. The Spearman correlation coefficients between the gene-level ribosome occupancies are indicated on the top left corner.

    Techniques Used: Nucleic Acid Electrophoresis, Labeling, Selection, Marker, Sequencing, Isolation

    12) Product Images from "Mechanistic insights on the mode of action of an antiproliferative thiosemicarbazone-nickel complex revealed by an integrated chemogenomic profiling study"

    Article Title: Mechanistic insights on the mode of action of an antiproliferative thiosemicarbazone-nickel complex revealed by an integrated chemogenomic profiling study

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-67439-y

    Ni(S-tcitr) 2 affects chromatin remodeling and microtubule cytoskeleton functionality. ( a ) Effect of Ni(S-tcitr) 2 treatment on in vivo chromatin remodeling. Equal amounts of spheroplasts from yeast cells treated with Ni(S-tcitr) 2 or DMSO (vehicle) were subjected to chromatin digestion with micrococcal nuclease (MNase); purified DNA was then fractionated by agarose gel electrophoresis and visualized by ethidium bromide staining (see ‘ Methods ’ for details). A representative gel picture shows different patterns of MNase digestion products. ( b,c ) Relative quantification of nucleosome-size fragments obtained from digestion of Ni(S-tcitr) 2 -treated and control (DMSO) chromatin samples with high (10 U; b ) or low (2.5 U; c ) amounts of MNase. ( d ) Synergistic toxicity of Ni(S-tcitr) 2 and the antimicrotubule drug benomyl. Ten-fold serial dilutions of wild-type (WT) cells and yeast mutant strains deleted in genes coding for chromatin remodeling ( arp6Δ and arp8Δ ), microtubule ( alf1Δ ) or cell cycle checkpoint components ( bub1Δ ) were plated onto YPD agar plates containing sublethal concentrations of benomyl (17 µM) and /or Ni(S-tcitr) 2 (10 µM) as indicated, and incubated for 2 days at 28 °C.
    Figure Legend Snippet: Ni(S-tcitr) 2 affects chromatin remodeling and microtubule cytoskeleton functionality. ( a ) Effect of Ni(S-tcitr) 2 treatment on in vivo chromatin remodeling. Equal amounts of spheroplasts from yeast cells treated with Ni(S-tcitr) 2 or DMSO (vehicle) were subjected to chromatin digestion with micrococcal nuclease (MNase); purified DNA was then fractionated by agarose gel electrophoresis and visualized by ethidium bromide staining (see ‘ Methods ’ for details). A representative gel picture shows different patterns of MNase digestion products. ( b,c ) Relative quantification of nucleosome-size fragments obtained from digestion of Ni(S-tcitr) 2 -treated and control (DMSO) chromatin samples with high (10 U; b ) or low (2.5 U; c ) amounts of MNase. ( d ) Synergistic toxicity of Ni(S-tcitr) 2 and the antimicrotubule drug benomyl. Ten-fold serial dilutions of wild-type (WT) cells and yeast mutant strains deleted in genes coding for chromatin remodeling ( arp6Δ and arp8Δ ), microtubule ( alf1Δ ) or cell cycle checkpoint components ( bub1Δ ) were plated onto YPD agar plates containing sublethal concentrations of benomyl (17 µM) and /or Ni(S-tcitr) 2 (10 µM) as indicated, and incubated for 2 days at 28 °C.

    Techniques Used: In Vivo, Purification, Agarose Gel Electrophoresis, Staining, Mutagenesis, Incubation

    13) Product Images from "Atomic-resolution mapping of transcription factor-DNA interactions by femtosecond laser crosslinking and mass spectrometry"

    Article Title: Atomic-resolution mapping of transcription factor-DNA interactions by femtosecond laser crosslinking and mass spectrometry

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16837-x

    Schematic workflow of the fliX-MS pipeline. a A pulsed laser beam was generated using a femtosecond fiber laser with 515 nm wavelength, repetition rate of 0.5 MHz, and pulse duration of 500 fs. The wavelength was doubled to 258 nm by second harmonic generation (SHG) over a beta barium borate (BBO) crystal and the laser beam adjusted to fit the inner diameter of a regular 1.5 ml Eppendorf tube. b Protein–DNA complexes were irradiated or left untreated as control. Samples were denatured, DNA digested to mono/short oligonucleotides by a mix of Mnase, DNase I, and Benzonase, and proteins digested to peptides by trypsin and Lys-C. Peptides and peptide–nucleotide cross-links were separated from free DNA on C18 StageTips 25 , and cross-links subsequently enriched with TiO 2 beads. c Peptides were measured by LC–MS/MS and data analyzed with the RNP(xl) software package implemented in the proteome discoverer software 50 followed by manual annotation of candidate spectra.
    Figure Legend Snippet: Schematic workflow of the fliX-MS pipeline. a A pulsed laser beam was generated using a femtosecond fiber laser with 515 nm wavelength, repetition rate of 0.5 MHz, and pulse duration of 500 fs. The wavelength was doubled to 258 nm by second harmonic generation (SHG) over a beta barium borate (BBO) crystal and the laser beam adjusted to fit the inner diameter of a regular 1.5 ml Eppendorf tube. b Protein–DNA complexes were irradiated or left untreated as control. Samples were denatured, DNA digested to mono/short oligonucleotides by a mix of Mnase, DNase I, and Benzonase, and proteins digested to peptides by trypsin and Lys-C. Peptides and peptide–nucleotide cross-links were separated from free DNA on C18 StageTips 25 , and cross-links subsequently enriched with TiO 2 beads. c Peptides were measured by LC–MS/MS and data analyzed with the RNP(xl) software package implemented in the proteome discoverer software 50 followed by manual annotation of candidate spectra.

    Techniques Used: Generated, Irradiation, Liquid Chromatography with Mass Spectroscopy, Software

    14) Product Images from "Fundamental Role Of The H2A.Z C-Terminal Tail In The Formation Of Constitutive Heterochromatin"

    Article Title: Fundamental Role Of The H2A.Z C-Terminal Tail In The Formation Of Constitutive Heterochromatin

    Journal: bioRxiv

    doi: 10.1101/2021.02.22.432230

    Global differences in chromatin accessibility features between DKO H2A.Z1ΔC and H2A.Z1 nuclei. (A and B) Increased amount of intercalated EBr (A) and SYBRGold (B) was measured in H2A.ZΔC (ΔC) as compared to H2A.Z1 (CTRL) by flow-cytometric analyzes. Box-and-whisker plot was created from the mean fluorescence intensities of 4 parallel measurements. (C) and (D) Comparison of the sensitivity of chromatin to MNase (C) and DNase I (D). The DNA content of H2A.Z1ΔC (ΔC) and H2A.Z1 (CTRL) nuclei was measured by LSC after endonuclease treatment. (E-G) Comparison of the sensitivity of chromatin to a nickase. Halo size (E and F) and DNA content (G) of H2A.Z1ΔC (ΔC) and H2A.Z1 (CTRL) nuclei were measured by LSC after 0.5 U/ml nickase treatment.
    Figure Legend Snippet: Global differences in chromatin accessibility features between DKO H2A.Z1ΔC and H2A.Z1 nuclei. (A and B) Increased amount of intercalated EBr (A) and SYBRGold (B) was measured in H2A.ZΔC (ΔC) as compared to H2A.Z1 (CTRL) by flow-cytometric analyzes. Box-and-whisker plot was created from the mean fluorescence intensities of 4 parallel measurements. (C) and (D) Comparison of the sensitivity of chromatin to MNase (C) and DNase I (D). The DNA content of H2A.Z1ΔC (ΔC) and H2A.Z1 (CTRL) nuclei was measured by LSC after endonuclease treatment. (E-G) Comparison of the sensitivity of chromatin to a nickase. Halo size (E and F) and DNA content (G) of H2A.Z1ΔC (ΔC) and H2A.Z1 (CTRL) nuclei were measured by LSC after 0.5 U/ml nickase treatment.

    Techniques Used: Whisker Assay, Fluorescence

    15) Product Images from "Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky526

    Dynamics of H2B K34ub nucleosomes. ( A ) Nucleosomes assembled on 147 bp 601 DNA were resolved in native PAGE under ionic and temperature conditions indicated on top. For the middle panel, although electrophoresis was performed at ∼26°C, the temperature at the glass surface was ∼35–37°C due to higher conductivity of the buffer. All gels were pre-electrophoresed in the relevant buffer. ( B ) Unmodified and H2B K34ub nucleosomes /hexasomes assembled on 177 bp 601 were digested with MNase at 26°C or 37°C and DNA was resolved in 6.5% PAGE and stained with SYBR Gold.
    Figure Legend Snippet: Dynamics of H2B K34ub nucleosomes. ( A ) Nucleosomes assembled on 147 bp 601 DNA were resolved in native PAGE under ionic and temperature conditions indicated on top. For the middle panel, although electrophoresis was performed at ∼26°C, the temperature at the glass surface was ∼35–37°C due to higher conductivity of the buffer. All gels were pre-electrophoresed in the relevant buffer. ( B ) Unmodified and H2B K34ub nucleosomes /hexasomes assembled on 177 bp 601 were digested with MNase at 26°C or 37°C and DNA was resolved in 6.5% PAGE and stained with SYBR Gold.

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

    16) Product Images from "CAL1 is the Drosophila CENP-A assembly factor"

    Article Title: CAL1 is the Drosophila CENP-A assembly factor

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201305036

    CAL1 is a CENP-A–specific nucleosome assembly factor. (A) CENP-A 101–225 –H4 was assembled on circular relaxed plasmid (pCR2.1-4 [CEN3 + CEN6]) using increasing amounts of CAL1 1–96 , CAL1 1–132 , or CAL1 1–160 in the presence of topoisomerase I. The extracted DNA samples were analyzed on agarose gel and stained with SYBR gold. Lane 1: supercoiled plasmid; lane 2: relaxed plasmid; lane 3: CENP-A 101–225 –H4, H2A–H2B but no CAL1; lanes 4–7: relaxed plasmid and CENP-A 101–225 –H4, H2A–H2B with CAL1 1–96 ; lanes 8–11: CENP-A 101–225 –H4, H2A–H2B with CAL1 1–132 ; lanes 12–15: CENP-A 101–225 –H4, H2A–H2B with CAL1 1–160 . (B) CENP-A–containing nucleosomes and histone H3 nucleosomes were assembled using histone proteins, CAL1 1–160 , or yeast Nap1 and pGEM3Z-601 plasmid. Lane 1: supercoiled plasmid; lane 2: relaxed plasmid; lanes 3, 6, and 9: mock reaction without CAL1 1–160 or Nap1; lanes 4 and 5: H3–H4, H2A–H2B with CAL1 1–160 ; lanes 7 and 8: CENP-A 101–225 –H4, H2A-H2B with CAL1 1–160 ; lanes 10 and 11: H3–H4, H2A–H2B with Nap1; lanes 12 and 13: CENP-A 101–225 –H4, H2A–H2B with Nap1. (C) CAL1-assembled CENP-A nucleosomes are negatively supercoiled. CENP-A nucleosomes (lane CENP-A) were assembled by CAL1 1–160 in the presence of topoisomerase I and H2A–H2B dimers, whereas histone H3 nucleosomes (lane H3) were assembled by Nap1. Controls are: supercoiled plasmid (superc.), relaxed plasmid (relaxed), and relaxed plasmid without histones or Nap1, but treated and processed as in the H3 and CENP-A nucleosome assembly reactions (mock). The samples were analyzed on agarose gels with or without 1 µg/ml chloroquine. Boxes highlight the decrease in migration that occurs in the presence of chloroquine in both H3 and CENP-A nucleosomes. (A–C) S, supercoiled; R, relaxed plasmid. (D) Diagram depicting the expected migration patterns for negatively and positively supercoiled DNA separated by 2D gel electrophoresis ( Tachiwana et al., 2011 ). Left gel: H3 nucleosomes assembled with Nap1; right gel: CENP-A nucleosomes assembled with CAL1 1–160 . (E) Mononucleosomes were assembled on 147-bp Widom DNA with Nap1 or CAL1 1–160 and digested with MNase for 30 or 120 s. Native PAGE of samples shown. Lanes 1–3: DNA only; lanes 4 and 5: H3 nucleosomes assembled by Nap1; lanes 6 and 7: CENP-A nucleosomes assembled by Nap1; lanes 8 and 9: CENP-A nucleosomes assembled by CAL1 1–160 .
    Figure Legend Snippet: CAL1 is a CENP-A–specific nucleosome assembly factor. (A) CENP-A 101–225 –H4 was assembled on circular relaxed plasmid (pCR2.1-4 [CEN3 + CEN6]) using increasing amounts of CAL1 1–96 , CAL1 1–132 , or CAL1 1–160 in the presence of topoisomerase I. The extracted DNA samples were analyzed on agarose gel and stained with SYBR gold. Lane 1: supercoiled plasmid; lane 2: relaxed plasmid; lane 3: CENP-A 101–225 –H4, H2A–H2B but no CAL1; lanes 4–7: relaxed plasmid and CENP-A 101–225 –H4, H2A–H2B with CAL1 1–96 ; lanes 8–11: CENP-A 101–225 –H4, H2A–H2B with CAL1 1–132 ; lanes 12–15: CENP-A 101–225 –H4, H2A–H2B with CAL1 1–160 . (B) CENP-A–containing nucleosomes and histone H3 nucleosomes were assembled using histone proteins, CAL1 1–160 , or yeast Nap1 and pGEM3Z-601 plasmid. Lane 1: supercoiled plasmid; lane 2: relaxed plasmid; lanes 3, 6, and 9: mock reaction without CAL1 1–160 or Nap1; lanes 4 and 5: H3–H4, H2A–H2B with CAL1 1–160 ; lanes 7 and 8: CENP-A 101–225 –H4, H2A-H2B with CAL1 1–160 ; lanes 10 and 11: H3–H4, H2A–H2B with Nap1; lanes 12 and 13: CENP-A 101–225 –H4, H2A–H2B with Nap1. (C) CAL1-assembled CENP-A nucleosomes are negatively supercoiled. CENP-A nucleosomes (lane CENP-A) were assembled by CAL1 1–160 in the presence of topoisomerase I and H2A–H2B dimers, whereas histone H3 nucleosomes (lane H3) were assembled by Nap1. Controls are: supercoiled plasmid (superc.), relaxed plasmid (relaxed), and relaxed plasmid without histones or Nap1, but treated and processed as in the H3 and CENP-A nucleosome assembly reactions (mock). The samples were analyzed on agarose gels with or without 1 µg/ml chloroquine. Boxes highlight the decrease in migration that occurs in the presence of chloroquine in both H3 and CENP-A nucleosomes. (A–C) S, supercoiled; R, relaxed plasmid. (D) Diagram depicting the expected migration patterns for negatively and positively supercoiled DNA separated by 2D gel electrophoresis ( Tachiwana et al., 2011 ). Left gel: H3 nucleosomes assembled with Nap1; right gel: CENP-A nucleosomes assembled with CAL1 1–160 . (E) Mononucleosomes were assembled on 147-bp Widom DNA with Nap1 or CAL1 1–160 and digested with MNase for 30 or 120 s. Native PAGE of samples shown. Lanes 1–3: DNA only; lanes 4 and 5: H3 nucleosomes assembled by Nap1; lanes 6 and 7: CENP-A nucleosomes assembled by Nap1; lanes 8 and 9: CENP-A nucleosomes assembled by CAL1 1–160 .

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Staining, Migration, Two-Dimensional Gel Electrophoresis, Electrophoresis, Clear Native PAGE

    17) Product Images from "Identification of a DNA N6-adenine methyltransferase complex and its impact on chromatin organization"

    Article Title: Identification of a DNA N6-adenine methyltransferase complex and its impact on chromatin organization

    Journal: Cell

    doi: 10.1016/j.cell.2019.04.028

    Quantitative modulation of nucleosome occupancy by 6mA (A) Experimental workflow. Chromatin is assembled using either salt dialysis or the NAP1 histone chaperone. Italicized blue steps are selectively included. (B) Tiling qPCR analysis of synthetic chromosome with cognate 6mA sites. Horizontal grey box represents annotated gene, and vertical black lines depict native 6mA positions. Horizontal blue bars span ~100bp regions amplified by qPCR. Red horizontal lines represent the region containing 6mA. ‘Hemi methyl’ chromosomes contain 6mA on the antisense and sense strands, respectively, while the ‘Full methyl’ chromosome has 6mA on both strands. Black arrowheads: decrease in nucleosome occupancy specifically at the 6mA cluster. (C) Tiling qPCR analysis of ectopically methylated synthetic chromosome. Vertical black lines illustrate possible 6mA sites installed enzymatically. Red arrowheads: decrease in nucleosome occupancy in the ectopically methylated region. Black arrowheads: position of cognate 6mA sites (not in this construct). (D) Tiling qPCR analysis of chromatin from panel B that is subsequently incubated with ACF and/or ATP. ACF equalizes nucleosome occupancy between the 6mA cluster and flanking regions in the presence of ATP (black line). Nucleosome occupancy at the methylated region is not restored to the same level as the unmethylated control (black arrowheads). (E) MNase-seq analysis of chromatin is assembled on native gDNA (“+” 6mA) and mini-genome DNA (“−“ 6mA) using NAP1 +/− ACF and ATP. P -values were calculated using a two-sample unequal variance t-test.
    Figure Legend Snippet: Quantitative modulation of nucleosome occupancy by 6mA (A) Experimental workflow. Chromatin is assembled using either salt dialysis or the NAP1 histone chaperone. Italicized blue steps are selectively included. (B) Tiling qPCR analysis of synthetic chromosome with cognate 6mA sites. Horizontal grey box represents annotated gene, and vertical black lines depict native 6mA positions. Horizontal blue bars span ~100bp regions amplified by qPCR. Red horizontal lines represent the region containing 6mA. ‘Hemi methyl’ chromosomes contain 6mA on the antisense and sense strands, respectively, while the ‘Full methyl’ chromosome has 6mA on both strands. Black arrowheads: decrease in nucleosome occupancy specifically at the 6mA cluster. (C) Tiling qPCR analysis of ectopically methylated synthetic chromosome. Vertical black lines illustrate possible 6mA sites installed enzymatically. Red arrowheads: decrease in nucleosome occupancy in the ectopically methylated region. Black arrowheads: position of cognate 6mA sites (not in this construct). (D) Tiling qPCR analysis of chromatin from panel B that is subsequently incubated with ACF and/or ATP. ACF equalizes nucleosome occupancy between the 6mA cluster and flanking regions in the presence of ATP (black line). Nucleosome occupancy at the methylated region is not restored to the same level as the unmethylated control (black arrowheads). (E) MNase-seq analysis of chromatin is assembled on native gDNA (“+” 6mA) and mini-genome DNA (“−“ 6mA) using NAP1 +/− ACF and ATP. P -values were calculated using a two-sample unequal variance t-test.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Methylation, Construct, Incubation

    18) Product Images from "Identification of a DNA N6-adenine methyltransferase complex and its impact on chromatin organization"

    Article Title: Identification of a DNA N6-adenine methyltransferase complex and its impact on chromatin organization

    Journal: Cell

    doi: 10.1016/j.cell.2019.04.028

    Quantitative modulation of nucleosome occupancy by 6mA (A) Experimental workflow. Chromatin is assembled using either salt dialysis or the NAP1 histone chaperone. Italicized blue steps are selectively included. (B) Tiling qPCR analysis of synthetic chromosome with cognate 6mA sites. Horizontal grey box represents annotated gene, and vertical black lines depict native 6mA positions. Horizontal blue bars span ~100bp regions amplified by qPCR. Red horizontal lines represent the region containing 6mA. ‘Hemi methyl’ chromosomes contain 6mA on the antisense and sense strands, respectively, while the ‘Full methyl’ chromosome has 6mA on both strands. Black arrowheads: decrease in nucleosome occupancy specifically at the 6mA cluster. (C) Tiling qPCR analysis of ectopically methylated synthetic chromosome. Vertical black lines illustrate possible 6mA sites installed enzymatically. Red arrowheads: decrease in nucleosome occupancy in the ectopically methylated region. Black arrowheads: position of cognate 6mA sites (not in this construct). (D) Tiling qPCR analysis of chromatin from panel B that is subsequently incubated with ACF and/or ATP. ACF equalizes nucleosome occupancy between the 6mA cluster and flanking regions in the presence of ATP (black line). Nucleosome occupancy at the methylated region is not restored to the same level as the unmethylated control (black arrowheads). (E) MNase-seq analysis of chromatin is assembled on native gDNA (“+” 6mA) and mini-genome DNA (“−“ 6mA) using NAP1 +/− ACF and ATP. P -values were calculated using a two-sample unequal variance t-test.
    Figure Legend Snippet: Quantitative modulation of nucleosome occupancy by 6mA (A) Experimental workflow. Chromatin is assembled using either salt dialysis or the NAP1 histone chaperone. Italicized blue steps are selectively included. (B) Tiling qPCR analysis of synthetic chromosome with cognate 6mA sites. Horizontal grey box represents annotated gene, and vertical black lines depict native 6mA positions. Horizontal blue bars span ~100bp regions amplified by qPCR. Red horizontal lines represent the region containing 6mA. ‘Hemi methyl’ chromosomes contain 6mA on the antisense and sense strands, respectively, while the ‘Full methyl’ chromosome has 6mA on both strands. Black arrowheads: decrease in nucleosome occupancy specifically at the 6mA cluster. (C) Tiling qPCR analysis of ectopically methylated synthetic chromosome. Vertical black lines illustrate possible 6mA sites installed enzymatically. Red arrowheads: decrease in nucleosome occupancy in the ectopically methylated region. Black arrowheads: position of cognate 6mA sites (not in this construct). (D) Tiling qPCR analysis of chromatin from panel B that is subsequently incubated with ACF and/or ATP. ACF equalizes nucleosome occupancy between the 6mA cluster and flanking regions in the presence of ATP (black line). Nucleosome occupancy at the methylated region is not restored to the same level as the unmethylated control (black arrowheads). (E) MNase-seq analysis of chromatin is assembled on native gDNA (“+” 6mA) and mini-genome DNA (“−“ 6mA) using NAP1 +/− ACF and ATP. P -values were calculated using a two-sample unequal variance t-test.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Methylation, Construct, Incubation

    19) Product Images from "Mammalian SWI/SNF chromatin remodeler is essential for reductional meiosis in males"

    Article Title: Mammalian SWI/SNF chromatin remodeler is essential for reductional meiosis in males

    Journal: Nature Communications

    doi: 10.1038/s41467-021-26828-1

    ARID2 regulates chromatin organization in metaphase-I spermatocytes. a , b Control and Arid2 cKO metaphase-I spermatocyte squashes immunolabelled for PLK1 (green) and a H3T3P (magenta) and b H2AT120P (magenta). c , d Control and Arid2 cKO metaphase-I spermatocytes from cryosections treated with MNase (0–200 Kunitz units) c immunolabelled for PLK1 (green) and ARID2 (magenta), and d scored for chromatin accessibility indicated by the proportion of cells (horizontal axis) susceptible (accessible) or resistant (inaccessible) to MNase digestion at indicated MNase concentrations (vertical axis). The number of spermatocytes scored ( n ) are indicated. a – c DNA stained with DAPI. Scale bar: 5 μm, magnification: 100.8x. Int.Control: Internal control.
    Figure Legend Snippet: ARID2 regulates chromatin organization in metaphase-I spermatocytes. a , b Control and Arid2 cKO metaphase-I spermatocyte squashes immunolabelled for PLK1 (green) and a H3T3P (magenta) and b H2AT120P (magenta). c , d Control and Arid2 cKO metaphase-I spermatocytes from cryosections treated with MNase (0–200 Kunitz units) c immunolabelled for PLK1 (green) and ARID2 (magenta), and d scored for chromatin accessibility indicated by the proportion of cells (horizontal axis) susceptible (accessible) or resistant (inaccessible) to MNase digestion at indicated MNase concentrations (vertical axis). The number of spermatocytes scored ( n ) are indicated. a – c DNA stained with DAPI. Scale bar: 5 μm, magnification: 100.8x. Int.Control: Internal control.

    Techniques Used: Staining

    20) Product Images from "Integrating quantitative proteomics with accurate genome profiling of transcription factors by greenCUT RUN"

    Article Title: Integrating quantitative proteomics with accurate genome profiling of transcription factors by greenCUT RUN

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkab038

    Experimental approach of greenCUT RUN. Panels ( A and B ): Schematic of experimental strategy for greenCUT RUN (A) and CUT RUN (B). GreenCUT RUN is rapid and easy protocol, which involves only three steps to complete. Panel ( C ): The GST protein fused to the GFP nanobody and MNase (nanobody-MNase) was expressed and purified from bacteria. A single band of nanobody-MNase and protA-MNase were observed by coomassie staining of protein gels (panel C ). Panel ( D ): To access activity genomic DNA was treated with purified nanobody-MNase at different concentrations and compared with standard MNase and protA-MNase preparations. In panel D, lane 1 shows uncut genomic DNA, while lanes 2, 8 and 14 shows DNA after adding MNase in solution lacking Ca 2+ . Lanes 3–7, 9–13 and 15–19 shows DNA fragments after activating MNase with Ca 2+ . From lanes 3–7, 9–13 and 15–19, decreasing amount of MNases were used to access activity. Similar expression levels of endogenous and GFP-tagged TBP were observed. Panel ( E ): Expression of GFP-tagged NFYA, JUN, FOS and TBP after doxycycline induction as detected by GFP antibodies (left). Comparison of expression levels of GFP-tagged TBP with endogenous TBP (right). Panel ( F ): Volcano plot of GFP-tagged NFYA. Panel ( G ): Relative stoichiometries of top 20 proteins. In the stoichiometry calculation, NFYC is used for normalization.
    Figure Legend Snippet: Experimental approach of greenCUT RUN. Panels ( A and B ): Schematic of experimental strategy for greenCUT RUN (A) and CUT RUN (B). GreenCUT RUN is rapid and easy protocol, which involves only three steps to complete. Panel ( C ): The GST protein fused to the GFP nanobody and MNase (nanobody-MNase) was expressed and purified from bacteria. A single band of nanobody-MNase and protA-MNase were observed by coomassie staining of protein gels (panel C ). Panel ( D ): To access activity genomic DNA was treated with purified nanobody-MNase at different concentrations and compared with standard MNase and protA-MNase preparations. In panel D, lane 1 shows uncut genomic DNA, while lanes 2, 8 and 14 shows DNA after adding MNase in solution lacking Ca 2+ . Lanes 3–7, 9–13 and 15–19 shows DNA fragments after activating MNase with Ca 2+ . From lanes 3–7, 9–13 and 15–19, decreasing amount of MNases were used to access activity. Similar expression levels of endogenous and GFP-tagged TBP were observed. Panel ( E ): Expression of GFP-tagged NFYA, JUN, FOS and TBP after doxycycline induction as detected by GFP antibodies (left). Comparison of expression levels of GFP-tagged TBP with endogenous TBP (right). Panel ( F ): Volcano plot of GFP-tagged NFYA. Panel ( G ): Relative stoichiometries of top 20 proteins. In the stoichiometry calculation, NFYC is used for normalization.

    Techniques Used: Purification, Staining, Activity Assay, Expressing

    21) Product Images from "RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF"

    Article Title: RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky562

    Differential chromatin structure and transcription factor binding between consensus and Alu RBPJ binding sites. ( A ) Heat map of clustered reads densities for the indicated genome-wide determination or DNA sequence feature centered around RBPJ peak summits for all 28 220 RBPJ binding sites. ( B ) The same analysis as in A for the 4921 RBPJ peak summits that intersected with an Alu element within 200 bp. ( C ) Quantification of DNA ends densities for MNAse digested input, RBPJ immunoprecipitated, and DNase hypersensitive DNA for cluster 1 (without Alu) and cluster 5 (with Alu) regions.
    Figure Legend Snippet: Differential chromatin structure and transcription factor binding between consensus and Alu RBPJ binding sites. ( A ) Heat map of clustered reads densities for the indicated genome-wide determination or DNA sequence feature centered around RBPJ peak summits for all 28 220 RBPJ binding sites. ( B ) The same analysis as in A for the 4921 RBPJ peak summits that intersected with an Alu element within 200 bp. ( C ) Quantification of DNA ends densities for MNAse digested input, RBPJ immunoprecipitated, and DNase hypersensitive DNA for cluster 1 (without Alu) and cluster 5 (with Alu) regions.

    Techniques Used: Binding Assay, Genome Wide, Sequencing, Immunoprecipitation

    22) Product Images from "Synthesis of a eukaryotic chromosome reveals a role for N6-methyladenine in nucleosome organization"

    Article Title: Synthesis of a eukaryotic chromosome reveals a role for N6-methyladenine in nucleosome organization

    Journal: bioRxiv

    doi: 10.1101/184929

    Epigenomic profiles of chromatin, transcription and DNA methylation in Oxytricha chromosomes (A) Meta-chromosome plots overlaying MNase-seq (nucleosome occupancy in vivo ), SMRT-seq (m 6 dA), and 5’-complete cDNA sequencing data (transcription start sites; TSSs) at Oxytricha chromosome ends. Heterodimeric telomere protein complexes protect each end in vivo , and are denoted as orange ovals. The 5’ end is designated as being proximal to TSSs. (B) Frequency of m 6 dA modifications downstream of individual TSSs. Histogram plots denote the distribution of m 6 dA frequencies within each aggregate cluster. (C) Transcriptional activity is positively correlated with the total number of m 6 dA within the corresponding chromosome. RNAseq data are derived from poly(A)-enriched RNA. RPKM denotes the number of reads per kilobase of chromosome per million mapped reads. (D) Composite analysis of 65,107 methylation sites reveals that m 6 dA occurs within an 5’-ApT-3’ dinucleotide motif. (E) Distribution of various m 6 dA dinucleotide motifs across the genome. Asterisk indicates DNA N6-methyladenine.
    Figure Legend Snippet: Epigenomic profiles of chromatin, transcription and DNA methylation in Oxytricha chromosomes (A) Meta-chromosome plots overlaying MNase-seq (nucleosome occupancy in vivo ), SMRT-seq (m 6 dA), and 5’-complete cDNA sequencing data (transcription start sites; TSSs) at Oxytricha chromosome ends. Heterodimeric telomere protein complexes protect each end in vivo , and are denoted as orange ovals. The 5’ end is designated as being proximal to TSSs. (B) Frequency of m 6 dA modifications downstream of individual TSSs. Histogram plots denote the distribution of m 6 dA frequencies within each aggregate cluster. (C) Transcriptional activity is positively correlated with the total number of m 6 dA within the corresponding chromosome. RNAseq data are derived from poly(A)-enriched RNA. RPKM denotes the number of reads per kilobase of chromosome per million mapped reads. (D) Composite analysis of 65,107 methylation sites reveals that m 6 dA occurs within an 5’-ApT-3’ dinucleotide motif. (E) Distribution of various m 6 dA dinucleotide motifs across the genome. Asterisk indicates DNA N6-methyladenine.

    Techniques Used: DNA Methylation Assay, In Vivo, Sequencing, Activity Assay, Derivative Assay, Methylation

    m 6 dA shapes nucleosome organization across the genome (A) Experimental workflow. (B) Agarose gel analysis of Oxytricha gDNA (‘Native’) and mini-genome DNA before chromatin assembly. (C) Methylated regions of the genome exhibit lower nucleosome occupancy compared to identical DNA sequences lacking m 6 dA. Nucleosome occupancy (in vitro MNase-seq) and m 6 dA IP-seq coverage was calculated within overlapping 51bp windows across the 98 assayed chromosomes. Windows were binned according to the number of m 6 dA residues within. The MNase-seq coverage from chromatinized gDNA was divided by the corresponding coverage from chromatinized mini-genome DNA to obtain the fold change in nucleosome occupancy in each window (“+” histones). Naked gDNA and mini-genome DNA were also MNase-digested, sequenced and analyzed in the same manner to control for MNase sequence preferences (“-” histones). P values were calculated using a two-sample unequal variance t-test. (D) Tracks of m 6 dA distribution and MNase-seq coverage reveal a reduction in nucleosome occupancy at methylated loci (black arrowheads). “+” m 6 dA refers to chromatin assembled on Oxytricha gDNA, while “-” m 6 dA denotes chromatin assembled on mini-genome DNA. Similar trends are observed for both Oxytricha and Xenopus histones.
    Figure Legend Snippet: m 6 dA shapes nucleosome organization across the genome (A) Experimental workflow. (B) Agarose gel analysis of Oxytricha gDNA (‘Native’) and mini-genome DNA before chromatin assembly. (C) Methylated regions of the genome exhibit lower nucleosome occupancy compared to identical DNA sequences lacking m 6 dA. Nucleosome occupancy (in vitro MNase-seq) and m 6 dA IP-seq coverage was calculated within overlapping 51bp windows across the 98 assayed chromosomes. Windows were binned according to the number of m 6 dA residues within. The MNase-seq coverage from chromatinized gDNA was divided by the corresponding coverage from chromatinized mini-genome DNA to obtain the fold change in nucleosome occupancy in each window (“+” histones). Naked gDNA and mini-genome DNA were also MNase-digested, sequenced and analyzed in the same manner to control for MNase sequence preferences (“-” histones). P values were calculated using a two-sample unequal variance t-test. (D) Tracks of m 6 dA distribution and MNase-seq coverage reveal a reduction in nucleosome occupancy at methylated loci (black arrowheads). “+” m 6 dA refers to chromatin assembled on Oxytricha gDNA, while “-” m 6 dA denotes chromatin assembled on mini-genome DNA. Similar trends are observed for both Oxytricha and Xenopus histones.

    Techniques Used: Agarose Gel Electrophoresis, Methylation, In Vitro, Sequencing

    23) Product Images from "Fundamental Role Of The H2A.Z C-Terminal Tail In The Formation Of Constitutive Heterochromatin"

    Article Title: Fundamental Role Of The H2A.Z C-Terminal Tail In The Formation Of Constitutive Heterochromatin

    Journal: bioRxiv

    doi: 10.1101/2021.02.22.432230

    Global differences in chromatin accessibility features between DKO H2A.Z1ΔC and H2A.Z1 nuclei. (A and B) Increased amount of intercalated EBr (A) and SYBRGold (B) was measured in H2A.ZΔC (ΔC) as compared to H2A.Z1 (CTRL) by flow-cytometric analyzes. Box-and-whisker plot was created from the mean fluorescence intensities of 4 parallel measurements. (C) and (D) Comparison of the sensitivity of chromatin to MNase (C) and DNase I (D). The DNA content of H2A.Z1ΔC (ΔC) and H2A.Z1 (CTRL) nuclei was measured by LSC after endonuclease treatment. (E-G) Comparison of the sensitivity of chromatin to a nickase. Halo size (E and F) and DNA content (G) of H2A.Z1ΔC (ΔC) and H2A.Z1 (CTRL) nuclei were measured by LSC after 0.5 U/ml nickase treatment.
    Figure Legend Snippet: Global differences in chromatin accessibility features between DKO H2A.Z1ΔC and H2A.Z1 nuclei. (A and B) Increased amount of intercalated EBr (A) and SYBRGold (B) was measured in H2A.ZΔC (ΔC) as compared to H2A.Z1 (CTRL) by flow-cytometric analyzes. Box-and-whisker plot was created from the mean fluorescence intensities of 4 parallel measurements. (C) and (D) Comparison of the sensitivity of chromatin to MNase (C) and DNase I (D). The DNA content of H2A.Z1ΔC (ΔC) and H2A.Z1 (CTRL) nuclei was measured by LSC after endonuclease treatment. (E-G) Comparison of the sensitivity of chromatin to a nickase. Halo size (E and F) and DNA content (G) of H2A.Z1ΔC (ΔC) and H2A.Z1 (CTRL) nuclei were measured by LSC after 0.5 U/ml nickase treatment.

    Techniques Used: Whisker Assay, Fluorescence

    24) Product Images from "Micromanipulation of prophase I chromosomes from mouse spermatocytes reveals high stiffness and gel-like chromatin organization"

    Article Title: Micromanipulation of prophase I chromosomes from mouse spermatocytes reveals high stiffness and gel-like chromatin organization

    Journal: bioRxiv

    doi: 10.1101/2020.08.31.276402

    Mitotic and meiotic chromosomes have a contiguous DNA connection, which is dissolved by 4 bp restriction enzymes, but only weakened by 6 bp restriction enzymes. Image pairs show pipette positions untreated (native isolated) chromosomes when relaxed and stretched (a) , and chromosomes following enzyme treatments (b-d) . Vertical blue lines mark positions of force pipettes. Force pipette deflection by pulling (horizontal blue lines) indicates mechanical connection; no movement (no horizontal blue line) indicates no mechanical connection. Red notches mark positions of stiff pipettes. Bars are 5 μm. ( b ) Both mitotic and meiotic chromosomes were weakened, but not fully digested after treatment with PvuII (cut sequence CAG ˅ CTG). ( c ) Both mitotic and meiotic chromosomes lost connectivity after treatment with AluI (cut sequence AG ˅ CT; for 1 of 4 trials meiotic chromosomes were not fully digested by AluI). ( d ) Both mitotic and meiotic chromosomes lost connectivity when treated with MNase (cleaves all DNA sequences). ( e ) Quantification of chromosome stretching elasticity after no treatment or after being treated with PvuII, AluI, and MNase. No treatment caused a 13 ± 4% weakening of mitotic chromosomes (N=10) and a 1 ± 4% weakening of meiotic chromosomes (N=10). PvuII treatment caused a 70 ± 8% reduction in stiffness for MEF chromosomes (N=4) and 70 ± 9% reduction in stiffness for meiotic chromosomes (N=4). One of four AluI treatments of meiotic chromosomes caused a 90% reduction in stiffness (rather than fully digesting), while AluI treatment digested 4 of 4 mitotic chromosomes. All MNase treatments caused full digestion of mitotic and meiotic chromosomes (N=4 in both cases). All averages are reported as mean value ± SEM. Bars are 5 μm.
    Figure Legend Snippet: Mitotic and meiotic chromosomes have a contiguous DNA connection, which is dissolved by 4 bp restriction enzymes, but only weakened by 6 bp restriction enzymes. Image pairs show pipette positions untreated (native isolated) chromosomes when relaxed and stretched (a) , and chromosomes following enzyme treatments (b-d) . Vertical blue lines mark positions of force pipettes. Force pipette deflection by pulling (horizontal blue lines) indicates mechanical connection; no movement (no horizontal blue line) indicates no mechanical connection. Red notches mark positions of stiff pipettes. Bars are 5 μm. ( b ) Both mitotic and meiotic chromosomes were weakened, but not fully digested after treatment with PvuII (cut sequence CAG ˅ CTG). ( c ) Both mitotic and meiotic chromosomes lost connectivity after treatment with AluI (cut sequence AG ˅ CT; for 1 of 4 trials meiotic chromosomes were not fully digested by AluI). ( d ) Both mitotic and meiotic chromosomes lost connectivity when treated with MNase (cleaves all DNA sequences). ( e ) Quantification of chromosome stretching elasticity after no treatment or after being treated with PvuII, AluI, and MNase. No treatment caused a 13 ± 4% weakening of mitotic chromosomes (N=10) and a 1 ± 4% weakening of meiotic chromosomes (N=10). PvuII treatment caused a 70 ± 8% reduction in stiffness for MEF chromosomes (N=4) and 70 ± 9% reduction in stiffness for meiotic chromosomes (N=4). One of four AluI treatments of meiotic chromosomes caused a 90% reduction in stiffness (rather than fully digesting), while AluI treatment digested 4 of 4 mitotic chromosomes. All MNase treatments caused full digestion of mitotic and meiotic chromosomes (N=4 in both cases). All averages are reported as mean value ± SEM. Bars are 5 μm.

    Techniques Used: Transferring, Isolation, Sequencing

    25) Product Images from "Integrating quantitative proteomics with accurate genome profiling of transcription factors by greenCUT RUN"

    Article Title: Integrating quantitative proteomics with accurate genome profiling of transcription factors by greenCUT RUN

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkab038

    Experimental approach of greenCUT RUN. Panels ( A and B ): Schematic of experimental strategy for greenCUT RUN (A) and CUT RUN (B). GreenCUT RUN is rapid and easy protocol, which involves only three steps to complete. Panel ( C ): The GST protein fused to the GFP nanobody and MNase (nanobody-MNase) was expressed and purified from bacteria. A single band of nanobody-MNase and protA-MNase were observed by coomassie staining of protein gels (panel C ). Panel ( D ): To access activity genomic DNA was treated with purified nanobody-MNase at different concentrations and compared with standard MNase and protA-MNase preparations. In panel D, lane 1 shows uncut genomic DNA, while lanes 2, 8 and 14 shows DNA after adding MNase in solution lacking Ca 2+ . Lanes 3–7, 9–13 and 15–19 shows DNA fragments after activating MNase with Ca 2+ . From lanes 3–7, 9–13 and 15–19, decreasing amount of MNases were used to access activity. Similar expression levels of endogenous and GFP-tagged TBP were observed. Panel ( E ): Expression of GFP-tagged NFYA, JUN, FOS and TBP after doxycycline induction as detected by GFP antibodies (left). Comparison of expression levels of GFP-tagged TBP with endogenous TBP (right). Panel ( F ): Volcano plot of GFP-tagged NFYA. Panel ( G ): Relative stoichiometries of top 20 proteins. In the stoichiometry calculation, NFYC is used for normalization.
    Figure Legend Snippet: Experimental approach of greenCUT RUN. Panels ( A and B ): Schematic of experimental strategy for greenCUT RUN (A) and CUT RUN (B). GreenCUT RUN is rapid and easy protocol, which involves only three steps to complete. Panel ( C ): The GST protein fused to the GFP nanobody and MNase (nanobody-MNase) was expressed and purified from bacteria. A single band of nanobody-MNase and protA-MNase were observed by coomassie staining of protein gels (panel C ). Panel ( D ): To access activity genomic DNA was treated with purified nanobody-MNase at different concentrations and compared with standard MNase and protA-MNase preparations. In panel D, lane 1 shows uncut genomic DNA, while lanes 2, 8 and 14 shows DNA after adding MNase in solution lacking Ca 2+ . Lanes 3–7, 9–13 and 15–19 shows DNA fragments after activating MNase with Ca 2+ . From lanes 3–7, 9–13 and 15–19, decreasing amount of MNases were used to access activity. Similar expression levels of endogenous and GFP-tagged TBP were observed. Panel ( E ): Expression of GFP-tagged NFYA, JUN, FOS and TBP after doxycycline induction as detected by GFP antibodies (left). Comparison of expression levels of GFP-tagged TBP with endogenous TBP (right). Panel ( F ): Volcano plot of GFP-tagged NFYA. Panel ( G ): Relative stoichiometries of top 20 proteins. In the stoichiometry calculation, NFYC is used for normalization.

    Techniques Used: Purification, Staining, Activity Assay, Expressing

    26) Product Images from "Micromanipulation of prophase I chromosomes from mouse spermatocytes reveals high stiffness and gel-like chromatin organization"

    Article Title: Micromanipulation of prophase I chromosomes from mouse spermatocytes reveals high stiffness and gel-like chromatin organization

    Journal: Communications Biology

    doi: 10.1038/s42003-020-01265-w

    Mitotic and meiotic chromosomes have a contiguous DNA connection, which is dissolved by 4 bp restriction enzymes, but only weakened by 6 bp restriction enzymes. Image pairs show pipette positions untreated (native isolated) chromosomes when relaxed and stretched ( a ), and chromosomes following enzyme treatments ( b – d ). Vertical blue lines mark positions of force pipettes. Force pipette deflection by pulling (horizontal blue lines) indicates mechanical connection; no movement (no horizontal blue line) indicates no mechanical connection. Red notches mark positions of stiff pipettes. Bars are 5 µm. b Both mitotic and meiotic chromosomes were weakened, but not fully digested after treatment with PvuII (cut sequence CAG˅CTG). c Both mitotic and meiotic chromosomes lost connectivity after treatment with AluI (cut sequence AG˅CT; for 1 of 4 trials meiotic chromosomes were not fully digested by AluI). d Both mitotic and meiotic chromosomes lost connectivity when treated with MNase (cleaves all DNA sequences). e Quantification of chromosome stretching elasticity after no treatment or after being treated with PvuII, AluI, and MNase. No treatment caused a 13 ± 4% weakening of mitotic chromosomes ( N = 10) and a 1 ± 4% weakening of meiotic chromosomes ( N = 10). PvuII treatment caused a 70 ± 8% reduction in stiffness for MEF chromosomes ( N = 4) and 70 ± 9% reduction in stiffness for meiotic chromosomes ( N = 4). One of four AluI treatments of meiotic chromosomes caused a 90% reduction in stiffness (rather than fully digesting), while AluI treatment digested 4 of 4 mitotic chromosomes. All MNase treatments caused full digestion of mitotic and meiotic chromosomes ( N = 4 in both cases). All averages are reported as mean value ± SEM. Bars are 5 µm.
    Figure Legend Snippet: Mitotic and meiotic chromosomes have a contiguous DNA connection, which is dissolved by 4 bp restriction enzymes, but only weakened by 6 bp restriction enzymes. Image pairs show pipette positions untreated (native isolated) chromosomes when relaxed and stretched ( a ), and chromosomes following enzyme treatments ( b – d ). Vertical blue lines mark positions of force pipettes. Force pipette deflection by pulling (horizontal blue lines) indicates mechanical connection; no movement (no horizontal blue line) indicates no mechanical connection. Red notches mark positions of stiff pipettes. Bars are 5 µm. b Both mitotic and meiotic chromosomes were weakened, but not fully digested after treatment with PvuII (cut sequence CAG˅CTG). c Both mitotic and meiotic chromosomes lost connectivity after treatment with AluI (cut sequence AG˅CT; for 1 of 4 trials meiotic chromosomes were not fully digested by AluI). d Both mitotic and meiotic chromosomes lost connectivity when treated with MNase (cleaves all DNA sequences). e Quantification of chromosome stretching elasticity after no treatment or after being treated with PvuII, AluI, and MNase. No treatment caused a 13 ± 4% weakening of mitotic chromosomes ( N = 10) and a 1 ± 4% weakening of meiotic chromosomes ( N = 10). PvuII treatment caused a 70 ± 8% reduction in stiffness for MEF chromosomes ( N = 4) and 70 ± 9% reduction in stiffness for meiotic chromosomes ( N = 4). One of four AluI treatments of meiotic chromosomes caused a 90% reduction in stiffness (rather than fully digesting), while AluI treatment digested 4 of 4 mitotic chromosomes. All MNase treatments caused full digestion of mitotic and meiotic chromosomes ( N = 4 in both cases). All averages are reported as mean value ± SEM. Bars are 5 µm.

    Techniques Used: Transferring, Isolation, Sequencing

    27) Product Images from "Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure"

    Article Title: Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku150

    Nucleosome duplexes are decoupled into mononucleosomes. ( A ) EMSA of purified nucleosome duplexes containing H3(T118ph) HO and mp2-187 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S4 ). ( B ) Quantification of fraction of nucleosome duplexes (squares), nucleosome (circles) and free DNA (diamond) species for the gel in (A) versus time. Error bars are the standard deviation of three independent experiments. ( C ) EMSA of purified nucleosome duplexes and altosomes containing H3(T118ph) HO and mp2-247 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert in part to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S5 ). ( D ) Quantification of the fraction of nucleosome duplexes (squares), altosomes (triangles), nucleosome (circles) and free DNA (diamond) species for the gel in (C) versus time. Error bars are the standard deviation of three independent experiments.
    Figure Legend Snippet: Nucleosome duplexes are decoupled into mononucleosomes. ( A ) EMSA of purified nucleosome duplexes containing H3(T118ph) HO and mp2-187 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S4 ). ( B ) Quantification of fraction of nucleosome duplexes (squares), nucleosome (circles) and free DNA (diamond) species for the gel in (A) versus time. Error bars are the standard deviation of three independent experiments. ( C ) EMSA of purified nucleosome duplexes and altosomes containing H3(T118ph) HO and mp2-247 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert in part to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S5 ). ( D ) Quantification of the fraction of nucleosome duplexes (squares), altosomes (triangles), nucleosome (circles) and free DNA (diamond) species for the gel in (C) versus time. Error bars are the standard deviation of three independent experiments.

    Techniques Used: Purification, Standard Deviation

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    New England Biolabs mnase
    Dynamics of H2B K34ub nucleosomes. ( A ) Nucleosomes assembled on 147 bp 601 <t>DNA</t> were resolved in native PAGE under ionic and temperature conditions indicated on top. For the middle panel, although electrophoresis was performed at ∼26°C, the temperature at the glass surface was ∼35–37°C due to higher conductivity of the buffer. All gels were pre-electrophoresed in the relevant buffer. ( B ) Unmodified and H2B K34ub nucleosomes /hexasomes assembled on 177 bp 601 were digested with <t>MNase</t> at 26°C or 37°C and DNA was resolved in 6.5% PAGE and stained with SYBR Gold.
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    Dynamics of H2B K34ub nucleosomes. ( A ) Nucleosomes assembled on 147 bp 601 DNA were resolved in native PAGE under ionic and temperature conditions indicated on top. For the middle panel, although electrophoresis was performed at ∼26°C, the temperature at the glass surface was ∼35–37°C due to higher conductivity of the buffer. All gels were pre-electrophoresed in the relevant buffer. ( B ) Unmodified and H2B K34ub nucleosomes /hexasomes assembled on 177 bp 601 were digested with MNase at 26°C or 37°C and DNA was resolved in 6.5% PAGE and stained with SYBR Gold.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gky526

    Figure Lengend Snippet: Dynamics of H2B K34ub nucleosomes. ( A ) Nucleosomes assembled on 147 bp 601 DNA were resolved in native PAGE under ionic and temperature conditions indicated on top. For the middle panel, although electrophoresis was performed at ∼26°C, the temperature at the glass surface was ∼35–37°C due to higher conductivity of the buffer. All gels were pre-electrophoresed in the relevant buffer. ( B ) Unmodified and H2B K34ub nucleosomes /hexasomes assembled on 177 bp 601 were digested with MNase at 26°C or 37°C and DNA was resolved in 6.5% PAGE and stained with SYBR Gold.

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

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

    Effects of 6mA on nucleosome organization in vitro and in vivo Experimental workflow for the generation of mini-genome DNA from native genomic DNA, and subsequent analysis by MNase-seq. Agarose gel analysis of Oxytricha gDNA (‘Native’) and mini-genome DNA before chromatin assembly. Methylated regions in the genome exhibit lower nucleosome occupancy in vitro but not in vivo . MNase-seq (nucleosome occupancy) and 6mA IP-seq coverage were calculated within overlapping 51bp windows across the 98 assayed chromosomes. Windows were binned according to the number of 6mA residues within. The in vitro MNase-seq coverage from chromatinized native gDNA (“+” 6mA) was divided by the corresponding coverage from chromatinized mini-genome DNA (“-“ 6mA) to obtain the fold change in nucleosome occupancy in each window (“+” histones). A subtraction was performed on these datasets to obtain the difference in nucleosome occupancy in vitro . Identical DNA sequences were compared for each calculation. Naked native gDNA and mini-genome DNA were also MNase-digested, sequenced and analyzed in the same manner, to control for MNase sequence preferences (“-“ histones). Nucleosome occupancy in vivo corresponds to MNase-seq coverage from wild type and mta1 mutant cells. P -values were calculated using a two-sample unequal variance t-test. N.S denotes “non-significant”, with p > 0.05. Tracks of 6mA distribution and MNase-seq coverage reveal a reduction in nucleosome occupancy at methylated loci in vitro (black arrowheads). 6mA “+” strand and “-“ strand tracks refer to SMRT-seq base calls from wild type genomic DNA. For the in vitro MNase-seq tracks, “+ 6mA” refers to chromatin assembled on Oxytricha gDNA, while “-6mA” denotes chromatin assembled on mini-genome DNA. Similar trends are observed for both Oxytricha and Xenopus histones. Vertical axis for SMRT-seq data denotes confidence score [-10 log(p-value)] of detection of the methylation event, while that for in vitro MNase-seq data denotes normalized read coverage. Tracks of 6mA distribution and MNase-seq coverage in vivo reveal no change in nucleosome occupancy in linker regions despite loss of 6mA in mta1 mutants. Vertical axis for SMRT-seq tracks denote confidence score [-10 log(p-value)] of detection of the methylation event, while that for in vivo MNase-seq tracks denote normalized read coverage.

    Journal: bioRxiv

    Article Title: Reconstitution of eukaryotic chromosomes and manipulation of DNA N6-methyladenine alters chromatin and gene expression

    doi: 10.1101/475384

    Figure Lengend Snippet: Effects of 6mA on nucleosome organization in vitro and in vivo Experimental workflow for the generation of mini-genome DNA from native genomic DNA, and subsequent analysis by MNase-seq. Agarose gel analysis of Oxytricha gDNA (‘Native’) and mini-genome DNA before chromatin assembly. Methylated regions in the genome exhibit lower nucleosome occupancy in vitro but not in vivo . MNase-seq (nucleosome occupancy) and 6mA IP-seq coverage were calculated within overlapping 51bp windows across the 98 assayed chromosomes. Windows were binned according to the number of 6mA residues within. The in vitro MNase-seq coverage from chromatinized native gDNA (“+” 6mA) was divided by the corresponding coverage from chromatinized mini-genome DNA (“-“ 6mA) to obtain the fold change in nucleosome occupancy in each window (“+” histones). A subtraction was performed on these datasets to obtain the difference in nucleosome occupancy in vitro . Identical DNA sequences were compared for each calculation. Naked native gDNA and mini-genome DNA were also MNase-digested, sequenced and analyzed in the same manner, to control for MNase sequence preferences (“-“ histones). Nucleosome occupancy in vivo corresponds to MNase-seq coverage from wild type and mta1 mutant cells. P -values were calculated using a two-sample unequal variance t-test. N.S denotes “non-significant”, with p > 0.05. Tracks of 6mA distribution and MNase-seq coverage reveal a reduction in nucleosome occupancy at methylated loci in vitro (black arrowheads). 6mA “+” strand and “-“ strand tracks refer to SMRT-seq base calls from wild type genomic DNA. For the in vitro MNase-seq tracks, “+ 6mA” refers to chromatin assembled on Oxytricha gDNA, while “-6mA” denotes chromatin assembled on mini-genome DNA. Similar trends are observed for both Oxytricha and Xenopus histones. Vertical axis for SMRT-seq data denotes confidence score [-10 log(p-value)] of detection of the methylation event, while that for in vitro MNase-seq data denotes normalized read coverage. Tracks of 6mA distribution and MNase-seq coverage in vivo reveal no change in nucleosome occupancy in linker regions despite loss of 6mA in mta1 mutants. Vertical axis for SMRT-seq tracks denote confidence score [-10 log(p-value)] of detection of the methylation event, while that for in vivo MNase-seq tracks denote normalized read coverage.

    Article Snippet: The assembled chromatin was then adjusted to 50mM Tris pH 7.9, 5mM CaCl2 and digested with MNase (New England Biolabs) to mainly mononucleosomal DNA as previously described ( ).

    Techniques: In Vitro, In Vivo, Agarose Gel Electrophoresis, Methylation, Sequencing, Mutagenesis

    Epigenomic profiles of chromatin, transcription and DNA methylation in Oxytricha chromosomes Meta-chromosome plots overlaying in vivo MNase-seq (nucleosome occupancy), SMRT-seq (6mA), and 5’-complete cDNA sequencing data (transcription start sites; TSSs) at Oxytricha macronuclear chromosome ends. Heterodimeric telomere end- binding protein complexes protect each end in vivo and are denoted as orange ovals. Horizontal red bar denotes the promoter. The 5’ chromosome end is designated as being proximal to TSSs. +1, +2, and +3 nucleosomes are labeled relative to the 5’ chromosome end. Vertical axis: “Nucleosome occupancy” denotes MNase-seq read coverage; “6mA” denotes total number of detected 6mA marks; “Transcription start sites” denotes total number of called TSSs. Histograms of the total number of 6mA marks within each linker in Oxytricha chromosomes. Distinct linkers are depicted as horizontal bold blue lines. Transcriptional activity is positively correlated with 6mA levels. RNAseq data are derived from poly(A)-enriched RNA. Genes are sorted into 8 groups, according to the total number of 6mA marks between 0 bp to 800 bp downstream of the TSS. FPKM = Fragments per Kilobase of transcript per Million mapped RNAseq reads. Notch in the boxplot denotes median, ends of boxplot denote first and third quartiles, upper whisker denotes third quantile + 1.5 × interquartile range, and lower whisker denotes data quartile 1 − 1.5 × interquartile range. Composite analysis of 65,107 methylation sites reveals that 6mA (marked with * ) occurs within an 5’-ApT-3’ dinucleotide motif. Distribution of various 6mA dinucleotide motifs across the genome. Asterisk indicates DNA N6-methyladenine.

    Journal: bioRxiv

    Article Title: Reconstitution of eukaryotic chromosomes and manipulation of DNA N6-methyladenine alters chromatin and gene expression

    doi: 10.1101/475384

    Figure Lengend Snippet: Epigenomic profiles of chromatin, transcription and DNA methylation in Oxytricha chromosomes Meta-chromosome plots overlaying in vivo MNase-seq (nucleosome occupancy), SMRT-seq (6mA), and 5’-complete cDNA sequencing data (transcription start sites; TSSs) at Oxytricha macronuclear chromosome ends. Heterodimeric telomere end- binding protein complexes protect each end in vivo and are denoted as orange ovals. Horizontal red bar denotes the promoter. The 5’ chromosome end is designated as being proximal to TSSs. +1, +2, and +3 nucleosomes are labeled relative to the 5’ chromosome end. Vertical axis: “Nucleosome occupancy” denotes MNase-seq read coverage; “6mA” denotes total number of detected 6mA marks; “Transcription start sites” denotes total number of called TSSs. Histograms of the total number of 6mA marks within each linker in Oxytricha chromosomes. Distinct linkers are depicted as horizontal bold blue lines. Transcriptional activity is positively correlated with 6mA levels. RNAseq data are derived from poly(A)-enriched RNA. Genes are sorted into 8 groups, according to the total number of 6mA marks between 0 bp to 800 bp downstream of the TSS. FPKM = Fragments per Kilobase of transcript per Million mapped RNAseq reads. Notch in the boxplot denotes median, ends of boxplot denote first and third quartiles, upper whisker denotes third quantile + 1.5 × interquartile range, and lower whisker denotes data quartile 1 − 1.5 × interquartile range. Composite analysis of 65,107 methylation sites reveals that 6mA (marked with * ) occurs within an 5’-ApT-3’ dinucleotide motif. Distribution of various 6mA dinucleotide motifs across the genome. Asterisk indicates DNA N6-methyladenine.

    Article Snippet: The assembled chromatin was then adjusted to 50mM Tris pH 7.9, 5mM CaCl2 and digested with MNase (New England Biolabs) to mainly mononucleosomal DNA as previously described ( ).

    Techniques: DNA Methylation Assay, In Vivo, Sequencing, Binding Assay, Labeling, Activity Assay, Derivative Assay, Whisker Assay, Methylation

    Quantitative modulation of nucleosome occupancy by 6mA in synthetic chromosomes Experimental workflow. Chromatin is assembled using either salt dialysis or the NAP1 histone chaperone. Italicized blue steps are selectively included. Tiling qPCR analysis of nucleosome occupancy in the synthetic chromosome Contig1781.0, with cognate 6mA sites. Horizontal grey box represents the annotated gene within Contig1781.0, with vertical grey lines depicting 6mA positions as obtained from Figure 5A. Horizontal blue bars span ~100bp regions amplified by qPCR primer pairs. Red horizontal lines and vertical bars represent the region containing 6mA. ‘Hemi methyl’ chromosomes contain 6mA on the antisense and sense strands, respectively, while the ‘Full methyl’ chromosome has 6mA on both strands. Nucleosome occupancy represents normalized qPCR signal at each locus (see Methods). For each qPCR locus, “difference” = nucleosome occupancy in methylated chromosome – nucleosome occupancy in “no methyl chromosome”; “fold change” = nucleosome occupancy in methylated chromosome / nucleosome occupancy in no methyl chromosome. Black arrowheads denote the decrease in nucleosome occupancy specifically at the 6mA cluster. Tiling qPCR analysis of nucleosome occupancy in synthetic chromosome Contig1781.0 with ectopic 6mA sites. Vertical black lines illustrate possible 6mA sites installed enzymatically. Red arrowheads denote the decrease in nucleosome occupancy in the ectopically methylated region, while black arrowheads denote the position of cognate 6mA sites (not in this construct). The chromatin remodeler ACF can partially overcome the effects of 6mA on nucleosome organization in an ATP-dependent manner. Chromatin is assembled by salt dialysis on synthetic chromosomes from panel B, and subsequently incubated with ACF and/or ATP. ACF equalizes nucleosome occupancy between the 6mA cluster and flanking regions in the presence of ATP (black line), resulting in a relative increase in nucleosome occupancy across the methylated region. Nucleosome occupancy at the methylated region is not restored to the same level as the unmethylated control in the presence of ACF and ATP (black arrowheads). (E) Chromatin is assembled on native gDNA (“+” 6mA) and mini-genome DNA (“-“ 6mA) using NAP1 in the presence of ACF and/or ATP, and subsequently analyzed using MNase-seq. Sliding window fold change in nucleosome occupancy between native gDNA and mini-genome DNA is calculated as in Figure 4C. ACF acts in an ATP-dependent manner to restore nucleosome occupancy in methylated DNA windows. P -values were calculated using a two-sample unequal variance t-test.

    Journal: bioRxiv

    Article Title: Reconstitution of eukaryotic chromosomes and manipulation of DNA N6-methyladenine alters chromatin and gene expression

    doi: 10.1101/475384

    Figure Lengend Snippet: Quantitative modulation of nucleosome occupancy by 6mA in synthetic chromosomes Experimental workflow. Chromatin is assembled using either salt dialysis or the NAP1 histone chaperone. Italicized blue steps are selectively included. Tiling qPCR analysis of nucleosome occupancy in the synthetic chromosome Contig1781.0, with cognate 6mA sites. Horizontal grey box represents the annotated gene within Contig1781.0, with vertical grey lines depicting 6mA positions as obtained from Figure 5A. Horizontal blue bars span ~100bp regions amplified by qPCR primer pairs. Red horizontal lines and vertical bars represent the region containing 6mA. ‘Hemi methyl’ chromosomes contain 6mA on the antisense and sense strands, respectively, while the ‘Full methyl’ chromosome has 6mA on both strands. Nucleosome occupancy represents normalized qPCR signal at each locus (see Methods). For each qPCR locus, “difference” = nucleosome occupancy in methylated chromosome – nucleosome occupancy in “no methyl chromosome”; “fold change” = nucleosome occupancy in methylated chromosome / nucleosome occupancy in no methyl chromosome. Black arrowheads denote the decrease in nucleosome occupancy specifically at the 6mA cluster. Tiling qPCR analysis of nucleosome occupancy in synthetic chromosome Contig1781.0 with ectopic 6mA sites. Vertical black lines illustrate possible 6mA sites installed enzymatically. Red arrowheads denote the decrease in nucleosome occupancy in the ectopically methylated region, while black arrowheads denote the position of cognate 6mA sites (not in this construct). The chromatin remodeler ACF can partially overcome the effects of 6mA on nucleosome organization in an ATP-dependent manner. Chromatin is assembled by salt dialysis on synthetic chromosomes from panel B, and subsequently incubated with ACF and/or ATP. ACF equalizes nucleosome occupancy between the 6mA cluster and flanking regions in the presence of ATP (black line), resulting in a relative increase in nucleosome occupancy across the methylated region. Nucleosome occupancy at the methylated region is not restored to the same level as the unmethylated control in the presence of ACF and ATP (black arrowheads). (E) Chromatin is assembled on native gDNA (“+” 6mA) and mini-genome DNA (“-“ 6mA) using NAP1 in the presence of ACF and/or ATP, and subsequently analyzed using MNase-seq. Sliding window fold change in nucleosome occupancy between native gDNA and mini-genome DNA is calculated as in Figure 4C. ACF acts in an ATP-dependent manner to restore nucleosome occupancy in methylated DNA windows. P -values were calculated using a two-sample unequal variance t-test.

    Article Snippet: The assembled chromatin was then adjusted to 50mM Tris pH 7.9, 5mM CaCl2 and digested with MNase (New England Biolabs) to mainly mononucleosomal DNA as previously described ( ).

    Techniques: Real-time Polymerase Chain Reaction, Amplification, Methylation, Construct, Incubation

    BEN-2 shows B cell-specific nucleosome occupancy, chromatin accessibility and enrichment for the H3K27ac active enhancer histone mark across a panel of B cell lines. A. Nucleosome occupancy at BEN-2 as measured by ChART-PCR with MNase digestion. Data was normalised to the inaccessible SFTPA2 gene promoter such that a value of 1.0 represents fully compacted nucleosomes, and lower values indicate less compacted nucleosomes. B. Chromatin accessibility at BEN-2 as measured by ChART-PCR with DNase I digestion. Data have been normalised to the inaccessible SFTPA2 gene promoter. C. H3K27ac enrichment at BEN-2 as determined by ChIP-qPCR using the percent input method. Grey bars indicate H3K27ac enrichment at the target locus, and black bars show enrichment using a non-specific IgG control antibody. All data are presented as mean ± SEM from at least 3 biological replicates.

    Journal: bioRxiv

    Article Title: Regulatory architecture of the RCA gene cluster captures an intragenic TAD boundary and enhancer elements in B cells

    doi: 10.1101/2020.02.16.941070

    Figure Lengend Snippet: BEN-2 shows B cell-specific nucleosome occupancy, chromatin accessibility and enrichment for the H3K27ac active enhancer histone mark across a panel of B cell lines. A. Nucleosome occupancy at BEN-2 as measured by ChART-PCR with MNase digestion. Data was normalised to the inaccessible SFTPA2 gene promoter such that a value of 1.0 represents fully compacted nucleosomes, and lower values indicate less compacted nucleosomes. B. Chromatin accessibility at BEN-2 as measured by ChART-PCR with DNase I digestion. Data have been normalised to the inaccessible SFTPA2 gene promoter. C. H3K27ac enrichment at BEN-2 as determined by ChIP-qPCR using the percent input method. Grey bars indicate H3K27ac enrichment at the target locus, and black bars show enrichment using a non-specific IgG control antibody. All data are presented as mean ± SEM from at least 3 biological replicates.

    Article Snippet: To assess nucleosome occupancy, 1000 Gel Units MNase (New England Biolabs) was used.

    Techniques: Polymerase Chain Reaction, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    DAM accessibility correlates with nucleosome positions . This figure shows the superimposition of average DAM accessibility versus average MNase accessibility around the transcription start site (TSS) of 3,904 strongly expressed C. elegans genes. The dotted red curve represents a moving average of the muscle profile with a sliding window of 400 nucleotides. Positioned H3K4me2/3 nucleosomes are represented by ovals above the picture of the generic gene. Numbers on the nucleosomes indicate their positions relative to the TSS. [NDR = nucleosome depleted region]

    Journal: BMC Genomics

    Article Title: Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans

    doi: 10.1186/1471-2164-11-465

    Figure Lengend Snippet: DAM accessibility correlates with nucleosome positions . This figure shows the superimposition of average DAM accessibility versus average MNase accessibility around the transcription start site (TSS) of 3,904 strongly expressed C. elegans genes. The dotted red curve represents a moving average of the muscle profile with a sliding window of 400 nucleotides. Positioned H3K4me2/3 nucleosomes are represented by ovals above the picture of the generic gene. Numbers on the nucleosomes indicate their positions relative to the TSS. [NDR = nucleosome depleted region]

    Article Snippet: Abbreviations DALEC: Direct Asymmetric Ligation End Capture; DAM: DNA Adenine Methyltransferase; GFP: Green Fluorescent Protein; H3K4me2/3: di- or tri-methylation of lysine 4 on histone H3; MNase: micrococcal nuclease; NDR: Nucleosome Depleted Region; NEB: New England Biolabs; SAGE: Serial Analysis of Gene Expression

    Techniques: