Structured Review

Illumina Inc mnase
Engineering nucleosomal positioning on synthetic <t>DNA</t> sequences. ( A ) Nucleosomal profile of the genomic regions of S. pombe and S. cerevisiae , where the artificial sequences were inserted (red arrowheads). The restriction sites for HindIII (H), BsmI (B), and PagI (P) and the localization of the hybridization probes (green) are indicated. ( B ) The <t>MNase</t> end-labeling analysis of the S. pombe sequence integrated in S. pombe generates a regular nucleosomal profile as predicted (colored ovals) (Seq-Sp/Sp). Insertion of the same sequence in S. cerevisiae (bracket) generates a different pattern (Seq-Sp/Sc). ( C ) The S. cerevisiae sequence generates a regular profile after integration in S. cerevisiae (Seq-Sc/Sc) but fails to position nucleosomes when integrated in the S. pombe genome (Seq-Sc/Sp, bracket). ( D ) MNase-seq occupancy maps of the S. pombe artificial sequence integrated in S. pombe (red) or in S. cerevisiae (black). ( E ) MNase-seq occupancy maps of the S. cerevisiae artificial sequence integrated in S. cerevisiae (red) or in S. pombe (black).
Mnase, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mnase/product/Illumina Inc
Average 92 stars, based on 5 article reviews
Price from $9.99 to $1999.99
mnase - by Bioz Stars, 2020-05
92/100 stars

Images

1) Product Images from "Nucleosomal signatures impose nucleosome positioning in coding and noncoding sequences in the genome"

Article Title: Nucleosomal signatures impose nucleosome positioning in coding and noncoding sequences in the genome

Journal: Genome Research

doi: 10.1101/gr.207241.116

Engineering nucleosomal positioning on synthetic DNA sequences. ( A ) Nucleosomal profile of the genomic regions of S. pombe and S. cerevisiae , where the artificial sequences were inserted (red arrowheads). The restriction sites for HindIII (H), BsmI (B), and PagI (P) and the localization of the hybridization probes (green) are indicated. ( B ) The MNase end-labeling analysis of the S. pombe sequence integrated in S. pombe generates a regular nucleosomal profile as predicted (colored ovals) (Seq-Sp/Sp). Insertion of the same sequence in S. cerevisiae (bracket) generates a different pattern (Seq-Sp/Sc). ( C ) The S. cerevisiae sequence generates a regular profile after integration in S. cerevisiae (Seq-Sc/Sc) but fails to position nucleosomes when integrated in the S. pombe genome (Seq-Sc/Sp, bracket). ( D ) MNase-seq occupancy maps of the S. pombe artificial sequence integrated in S. pombe (red) or in S. cerevisiae (black). ( E ) MNase-seq occupancy maps of the S. cerevisiae artificial sequence integrated in S. cerevisiae (red) or in S. pombe (black).
Figure Legend Snippet: Engineering nucleosomal positioning on synthetic DNA sequences. ( A ) Nucleosomal profile of the genomic regions of S. pombe and S. cerevisiae , where the artificial sequences were inserted (red arrowheads). The restriction sites for HindIII (H), BsmI (B), and PagI (P) and the localization of the hybridization probes (green) are indicated. ( B ) The MNase end-labeling analysis of the S. pombe sequence integrated in S. pombe generates a regular nucleosomal profile as predicted (colored ovals) (Seq-Sp/Sp). Insertion of the same sequence in S. cerevisiae (bracket) generates a different pattern (Seq-Sp/Sc). ( C ) The S. cerevisiae sequence generates a regular profile after integration in S. cerevisiae (Seq-Sc/Sc) but fails to position nucleosomes when integrated in the S. pombe genome (Seq-Sc/Sp, bracket). ( D ) MNase-seq occupancy maps of the S. pombe artificial sequence integrated in S. pombe (red) or in S. cerevisiae (black). ( E ) MNase-seq occupancy maps of the S. cerevisiae artificial sequence integrated in S. cerevisiae (red) or in S. pombe (black).

Techniques Used: Hybridization, End Labeling, Sequencing

2) Product Images from "Histone H1 Limits DNA Methylation in Neurospora crassa"

Article Title: Histone H1 Limits DNA Methylation in Neurospora crassa

Journal: G3: Genes|Genomes|Genetics

doi: 10.1534/g3.116.028324

H1 is not a major determinant of nucleosome size or positioning in Neurospora . (A) Wild-type or (B) Δ hH1 nuclei were isolated and treated with micrococcal nuclease (MNase) for the indicated times (min). DNA was purified, resolved on an agarose gel, and visualized by staining with Ethidium Bromide. Wild-type and Δ hH1 display similar digestion kinetics. (C) Metaplots depict the average sequencing depth on the + strand across all N. crassa genes obtained by sequencing mononucleosomal DNA from a 60 min MNase digest. Plots show data from two biological replicates each for WT and Δ hH1 . (D–H) All N. crassa genes were ranked by expression level and split into quintile groups, ranging from highest expression (Quintile 1) to lowest expression (Quintile 5). Metaplots depict the average sequencing coverage on the + strand for each group. Plots show data from two biological replicates each for WT and Δ hH1 . The legend in the top right panel corresponds to plots (C–H). For all plots, the coverage of + strand reads from an H1-3XFLAG ChIP-seq experiment is shown as a dashed line. ChIP-seq; Chromatin-immunoprecipitation combined with sequencing; WT, wild-type.
Figure Legend Snippet: H1 is not a major determinant of nucleosome size or positioning in Neurospora . (A) Wild-type or (B) Δ hH1 nuclei were isolated and treated with micrococcal nuclease (MNase) for the indicated times (min). DNA was purified, resolved on an agarose gel, and visualized by staining with Ethidium Bromide. Wild-type and Δ hH1 display similar digestion kinetics. (C) Metaplots depict the average sequencing depth on the + strand across all N. crassa genes obtained by sequencing mononucleosomal DNA from a 60 min MNase digest. Plots show data from two biological replicates each for WT and Δ hH1 . (D–H) All N. crassa genes were ranked by expression level and split into quintile groups, ranging from highest expression (Quintile 1) to lowest expression (Quintile 5). Metaplots depict the average sequencing coverage on the + strand for each group. Plots show data from two biological replicates each for WT and Δ hH1 . The legend in the top right panel corresponds to plots (C–H). For all plots, the coverage of + strand reads from an H1-3XFLAG ChIP-seq experiment is shown as a dashed line. ChIP-seq; Chromatin-immunoprecipitation combined with sequencing; WT, wild-type.

Techniques Used: Isolation, Purification, Agarose Gel Electrophoresis, Staining, Sequencing, Expressing, Chromatin Immunoprecipitation

Related Articles

In Vivo:

Article Title: Evidence against a genomic code for nucleosome positioning
Article Snippet: .. In particular, the use of MNase and Illumina sequencing introduces systematic errors in the measurements and results in overestimates of the similarity between in vivo and in vitro samples. .. Illumina sequencing shows systematic differences in DNA sequence coverage depending on base composition and causes artifactually high correlations between samples .

Amplification:

Article Title: RSC-dependent constructive and destructive interference between opposing arrays of phased nucleosomes in yeast
Article Snippet: .. Nuclei were prepared and digested to nucleosome core particles with MNase as described previously , except that gel-purified core particle DNA was ligated to Index PE adapters (Illumina) and amplified with Illumina Index primers using 15 cycles of PCR. ..

In Vitro:

Article Title: Evidence against a genomic code for nucleosome positioning
Article Snippet: .. In particular, the use of MNase and Illumina sequencing introduces systematic errors in the measurements and results in overestimates of the similarity between in vivo and in vitro samples. .. Illumina sequencing shows systematic differences in DNA sequence coverage depending on base composition and causes artifactually high correlations between samples .

Isolation:

Article Title: Separate Polycomb Response Elements control chromatin state and activation of the vestigial gene
Article Snippet: .. MNase was then activated and finally the cleaved DNA was isolated, subjected to Illumina paired-end sequencing, and mapped to the Drosophila dm6 genome assembly. ..

Article Title: Nucleosomal signatures impose nucleosome positioning in coding and noncoding sequences in the genome
Article Snippet: .. Briefly, we digested chromatin with MNase, isolated mononucleosomal DNA, and sequenced it using the Illumina paired-end protocol (see Methods). ..

Construct:

Article Title: Histone H1 Limits DNA Methylation in Neurospora crassa
Article Snippet: .. We next performed gel extractions to isolate the DNA band corresponding to mononucleosomes (∼150 bp) from samples digested with MNase for 20 or 60 min and constructed Illumina sequencing libraries. ..

Purification:

Article Title: Mutation Bias, rather than Binding Preference, Underlies the Nucleosome-Associated G+C% Variation in Eukaryotes
Article Snippet: .. In that study, the yeast S. cerevisiae and E. coli genomic DNA were mixed in a 3:1 mass ratio and then incubated with purified histones by salt dialysis or by using a purified system containing recombinant D rosophila melanogaster NAP-1 and ACF as assistant factors; after reconstitution, the assembled chromatin was digested with MNase, and the mononucleosomal DNA was subsequently purified and sequenced by Illumina Genome Analyzer. .. As a control, the same S. cerevisiae and E. coli genomic DNA was sonicated into fragments of comparable size as to the mononucleosomal DNA and sequenced by the same platform.

Sequencing:

Article Title: Evidence against a genomic code for nucleosome positioning
Article Snippet: .. In particular, the use of MNase and Illumina sequencing introduces systematic errors in the measurements and results in overestimates of the similarity between in vivo and in vitro samples. .. Illumina sequencing shows systematic differences in DNA sequence coverage depending on base composition and causes artifactually high correlations between samples .

Article Title: Histone H1 Limits DNA Methylation in Neurospora crassa
Article Snippet: .. We next performed gel extractions to isolate the DNA band corresponding to mononucleosomes (∼150 bp) from samples digested with MNase for 20 or 60 min and constructed Illumina sequencing libraries. ..

Article Title: Nucleosomal occupancy changes locally over key regulatory regions during cell differentiation and reprogramming
Article Snippet: .. The resulting fragments from each MNase concentration in the range were prepared individually for barcoded sequencing on an Illumina HiSeq instrument. .. Illumina HiSeq library preparation and sequencing Mononucleosome DNA (1 μg) was used for library preparation, with limited number of PCR amplification rounds , and genomic alignments of paired-end 50 bp reads were performed using Bowtie followed by further tag processing and filtering with the SPP workflow .

Article Title: Separate Polycomb Response Elements control chromatin state and activation of the vestigial gene
Article Snippet: .. MNase was then activated and finally the cleaved DNA was isolated, subjected to Illumina paired-end sequencing, and mapped to the Drosophila dm6 genome assembly. ..

Concentration Assay:

Article Title: Nucleosomal occupancy changes locally over key regulatory regions during cell differentiation and reprogramming
Article Snippet: .. The resulting fragments from each MNase concentration in the range were prepared individually for barcoded sequencing on an Illumina HiSeq instrument. .. Illumina HiSeq library preparation and sequencing Mononucleosome DNA (1 μg) was used for library preparation, with limited number of PCR amplification rounds , and genomic alignments of paired-end 50 bp reads were performed using Bowtie followed by further tag processing and filtering with the SPP workflow .

Incubation:

Article Title: Mutation Bias, rather than Binding Preference, Underlies the Nucleosome-Associated G+C% Variation in Eukaryotes
Article Snippet: .. In that study, the yeast S. cerevisiae and E. coli genomic DNA were mixed in a 3:1 mass ratio and then incubated with purified histones by salt dialysis or by using a purified system containing recombinant D rosophila melanogaster NAP-1 and ACF as assistant factors; after reconstitution, the assembled chromatin was digested with MNase, and the mononucleosomal DNA was subsequently purified and sequenced by Illumina Genome Analyzer. .. As a control, the same S. cerevisiae and E. coli genomic DNA was sonicated into fragments of comparable size as to the mononucleosomal DNA and sequenced by the same platform.

E. coli Genomic Assay:

Article Title: Mutation Bias, rather than Binding Preference, Underlies the Nucleosome-Associated G+C% Variation in Eukaryotes
Article Snippet: .. In that study, the yeast S. cerevisiae and E. coli genomic DNA were mixed in a 3:1 mass ratio and then incubated with purified histones by salt dialysis or by using a purified system containing recombinant D rosophila melanogaster NAP-1 and ACF as assistant factors; after reconstitution, the assembled chromatin was digested with MNase, and the mononucleosomal DNA was subsequently purified and sequenced by Illumina Genome Analyzer. .. As a control, the same S. cerevisiae and E. coli genomic DNA was sonicated into fragments of comparable size as to the mononucleosomal DNA and sequenced by the same platform.

Polymerase Chain Reaction:

Article Title: RSC-dependent constructive and destructive interference between opposing arrays of phased nucleosomes in yeast
Article Snippet: .. Nuclei were prepared and digested to nucleosome core particles with MNase as described previously , except that gel-purified core particle DNA was ligated to Index PE adapters (Illumina) and amplified with Illumina Index primers using 15 cycles of PCR. ..

Recombinant:

Article Title: Mutation Bias, rather than Binding Preference, Underlies the Nucleosome-Associated G+C% Variation in Eukaryotes
Article Snippet: .. In that study, the yeast S. cerevisiae and E. coli genomic DNA were mixed in a 3:1 mass ratio and then incubated with purified histones by salt dialysis or by using a purified system containing recombinant D rosophila melanogaster NAP-1 and ACF as assistant factors; after reconstitution, the assembled chromatin was digested with MNase, and the mononucleosomal DNA was subsequently purified and sequenced by Illumina Genome Analyzer. .. As a control, the same S. cerevisiae and E. coli genomic DNA was sonicated into fragments of comparable size as to the mononucleosomal DNA and sequenced by the same platform.

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 85
    Illumina Inc mnase protected fragments
    Disposition of <t>nucleosomes</t> and RSC at the GAL1/10 locus ( A) Schematic of the chromatin architecture found at the GAL1/10 ). Nucleosomes are represented by green ovals, and two kinds of hypersensitive sites (HS and hs) are indicated by hatched bars. The white oval represents the unknown factor(s) that protect the UASg . The numbers between the arrows show the length of each segment protected from <t>MNase</t> digestion, and the numbers at the bottom show the percent of the population protected. The transcription start sites of the GAL1 and GAL10 genes are indicated by grey bars and the beginnings of the ORFs by black bars. The four Gal4 sites comprising the UASg are in cyan. The TATA box in the GAL1 promoter lies approximately coincident with the rightmost site marked hs. (B) RSC binding to the GAL1/10 locus. Yeast bearing TAP-tagged RSC were cross-linked, sonicated, and the isolated chromatin digested with MNase to an extent that yielded primarily mononucleosomes. RSC bound DNA-fragments were isolated on IgG-beads. The purified DNA was then subjected to paired-end high throughput (Illumina) DNA sequencing. The resulting fragments were mapped to the S. cerevisiae genome to determine their sizes and positions. The number of fragments that cross any given base pair in the GAL1/10 locus is shown by the blue line (the fragment density). The data is represented as fold enrichment of RSC bound fragments over a random distribution. Cells were grown in glucose at 25°C, and the TAP-tag was added to the RSC subunit RSC8 . (C) Inactivation of the RSC DNA binding subunit Rsc3. Cells bearing the ts mutant RSC ( rsc3-1 ), also TAP-tagged, were grown in glucose at 25°C and then shifted to 37°C for various times as indicated. Cells were treated as described in (B) and the recovered DNA analyzed by QPCR. RSC binding is presented as the percentage immunoprecipitated. (D) RSC binding in the absence of Gal4. Cells deleted for gal4 and bearing TAP-tagged wt RSC were analyzed for RSC binding and MNase sensitivity as described in (C). RSC binding is shown as fold over a control locus in the PHO5 shows that the MNase protection pattern in and around the UASg is not altered by deletion of gal4 . (E) Gal4 and putative RSC binding sites in the UASg . The Gal4 binding sites are indicated in blue. Gal4 sites each bind a dimer of the protein, and each of the three strong binding sites has the sequence CGG-N 11 ) are indicated in red. The rightmost putative RSC site overlaps Gal4 binding site 4, which differs from the Gal4 consensus in one base pair as shown. The arrow indicates the site of truncation of the UASg in the mutant strain created for the experiments in (F). Sequences to the right of the arrow are deleted in the truncation mutant. (F) RSC binding to a wt and a truncated UASg . Cells bearing TAP-tagged RSC and grown in raffinose were probed as described in (C).
    Mnase Protected Fragments, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mnase protected fragments/product/Illumina Inc
    Average 85 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    mnase protected fragments - by Bioz Stars, 2020-05
    85/100 stars
      Buy from Supplier

    90
    Illumina Inc micrococcal nuclease mnase
    Genome-wide nucleosome positioning in Dictyostelium . ( A ) Normalized read midpoint frequency distributions of <t>MNase-protected</t> fragments (nucleosome dyads) of all 12,750 genes in growth-stage WT cells were aligned relative to their ATG codons. Peaks (arrows) correspond to dyad midpoints for globally phased nucleosomes in the 5′ region of intragenic <t>DNA,</t> and distances between mapped read peaks correspond to ∼170 bp NRL. The protein coding DNA sequence (cds) region is shaded. ( B ) Normalized read midpoint frequency distributions of all genes in growth-stage WT cells were aligned relative to their translational termination sites (stop codons). Peaks (arrows) in the mean normalized frequency distribution correspond to globally phased nucleosomes in the 3′ region of intragenic DNA. The protein cds region is shaded. ( C ) Normalized dyad read midpoint frequency distributions for WT chromatin (CHR; dotted line) (see A ) were adjusted for sequence mappability by dividing with equivalent control data from MNase-digested naked (protein free) WT DNA (DNA; red line) and replotted as the ratio (CHR/DNA; thick black line) within 1.2 kb of flanking chromatin relative to ATG sites of all 12,750 genes. An ∼170-bp nucleosome-depleted (“free”) region (NDR) is centered near the AT-rich regions of Dictyostelium TSS. Positioned nucleosomes upstream (+) and downstream (−) to the NDR are indicated by arrows. The protein cds region is shaded.
    Micrococcal Nuclease Mnase, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/micrococcal nuclease mnase/product/Illumina Inc
    Average 90 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    micrococcal nuclease mnase - by Bioz Stars, 2020-05
    90/100 stars
      Buy from Supplier

    85
    Illumina Inc mnase digested genomic
    ( A ) <t>DNA</t> from <t>MNase</t> digested chromatin fractions purified from wild-type reference yeast strain BY4742 and an isogenic Δcbf1 mutant and separated by agarose gel electrophoresis. The lanes marked: ‘Total’ and ‘Input’ show the DNA species purified from MNase digested cells before and after a brief centrifugation step (to remove high-molecular weight material; see ‘Materials and Methods’ section) respectively. The ‘Input’ fractions were used for sequencing. Marker sizes are shown in kilobases and bands corresponding to DNA from mono-, di- and tri-nucleosomes are indicated to the right of the gel. ( B ) End-to-end distances of paired sequence reads reflect the distribution of chromatin particle input DNA. Graph of the number of Bowtie-aligned ( 9 ) paired-end reads obtained by Illumina GAIIx sequencing of material shown in Figure 1 A ‘Input’ lanes versus paired-read end-to-end distance (Bowtie SAM format insert size value). The values shown on the x -axis indicate aligned sequences with the initial read mapping to the R/+/Crick strand of the reference genome. F/–/Watson strand reads show an identical distribution ( Supplementary Figure S1 ). Peaks at ∼150, 300 and 450 bp are marked and correspond to mono-, di- and tri-nucleosome DNA fractions respectively. ( C ) Paired read size class dyad frequencies describe a landscape of MNase-protected species surrounding protein-coding gene TSSs. Using wild-type cell data, cumulative dyad frequencies for 15-bp bins, centred on and surrounding the protein coding gene TSSs mapped by ( 12 ), were plotted for paired read size classes 36 and 50– 700 bp in 25-bp intervals as a surface graph. Dyad frequencies within each size class were normalized to the mean number of reads per bin for that size class in order to plot each data set on the same y -axis. Normalized cumulative frequency values > 1 are coloured yellow; values
    Mnase Digested Genomic, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mnase digested genomic/product/Illumina Inc
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mnase digested genomic - by Bioz Stars, 2020-05
    85/100 stars
      Buy from Supplier

    Image Search Results


    Disposition of nucleosomes and RSC at the GAL1/10 locus ( A) Schematic of the chromatin architecture found at the GAL1/10 ). Nucleosomes are represented by green ovals, and two kinds of hypersensitive sites (HS and hs) are indicated by hatched bars. The white oval represents the unknown factor(s) that protect the UASg . The numbers between the arrows show the length of each segment protected from MNase digestion, and the numbers at the bottom show the percent of the population protected. The transcription start sites of the GAL1 and GAL10 genes are indicated by grey bars and the beginnings of the ORFs by black bars. The four Gal4 sites comprising the UASg are in cyan. The TATA box in the GAL1 promoter lies approximately coincident with the rightmost site marked hs. (B) RSC binding to the GAL1/10 locus. Yeast bearing TAP-tagged RSC were cross-linked, sonicated, and the isolated chromatin digested with MNase to an extent that yielded primarily mononucleosomes. RSC bound DNA-fragments were isolated on IgG-beads. The purified DNA was then subjected to paired-end high throughput (Illumina) DNA sequencing. The resulting fragments were mapped to the S. cerevisiae genome to determine their sizes and positions. The number of fragments that cross any given base pair in the GAL1/10 locus is shown by the blue line (the fragment density). The data is represented as fold enrichment of RSC bound fragments over a random distribution. Cells were grown in glucose at 25°C, and the TAP-tag was added to the RSC subunit RSC8 . (C) Inactivation of the RSC DNA binding subunit Rsc3. Cells bearing the ts mutant RSC ( rsc3-1 ), also TAP-tagged, were grown in glucose at 25°C and then shifted to 37°C for various times as indicated. Cells were treated as described in (B) and the recovered DNA analyzed by QPCR. RSC binding is presented as the percentage immunoprecipitated. (D) RSC binding in the absence of Gal4. Cells deleted for gal4 and bearing TAP-tagged wt RSC were analyzed for RSC binding and MNase sensitivity as described in (C). RSC binding is shown as fold over a control locus in the PHO5 shows that the MNase protection pattern in and around the UASg is not altered by deletion of gal4 . (E) Gal4 and putative RSC binding sites in the UASg . The Gal4 binding sites are indicated in blue. Gal4 sites each bind a dimer of the protein, and each of the three strong binding sites has the sequence CGG-N 11 ) are indicated in red. The rightmost putative RSC site overlaps Gal4 binding site 4, which differs from the Gal4 consensus in one base pair as shown. The arrow indicates the site of truncation of the UASg in the mutant strain created for the experiments in (F). Sequences to the right of the arrow are deleted in the truncation mutant. (F) RSC binding to a wt and a truncated UASg . Cells bearing TAP-tagged RSC and grown in raffinose were probed as described in (C).

    Journal: Cell

    Article Title: A RSC/nucleosome complex determines chromatin architecture and facilitates activator binding

    doi: 10.1016/j.cell.2010.03.048

    Figure Lengend Snippet: Disposition of nucleosomes and RSC at the GAL1/10 locus ( A) Schematic of the chromatin architecture found at the GAL1/10 ). Nucleosomes are represented by green ovals, and two kinds of hypersensitive sites (HS and hs) are indicated by hatched bars. The white oval represents the unknown factor(s) that protect the UASg . The numbers between the arrows show the length of each segment protected from MNase digestion, and the numbers at the bottom show the percent of the population protected. The transcription start sites of the GAL1 and GAL10 genes are indicated by grey bars and the beginnings of the ORFs by black bars. The four Gal4 sites comprising the UASg are in cyan. The TATA box in the GAL1 promoter lies approximately coincident with the rightmost site marked hs. (B) RSC binding to the GAL1/10 locus. Yeast bearing TAP-tagged RSC were cross-linked, sonicated, and the isolated chromatin digested with MNase to an extent that yielded primarily mononucleosomes. RSC bound DNA-fragments were isolated on IgG-beads. The purified DNA was then subjected to paired-end high throughput (Illumina) DNA sequencing. The resulting fragments were mapped to the S. cerevisiae genome to determine their sizes and positions. The number of fragments that cross any given base pair in the GAL1/10 locus is shown by the blue line (the fragment density). The data is represented as fold enrichment of RSC bound fragments over a random distribution. Cells were grown in glucose at 25°C, and the TAP-tag was added to the RSC subunit RSC8 . (C) Inactivation of the RSC DNA binding subunit Rsc3. Cells bearing the ts mutant RSC ( rsc3-1 ), also TAP-tagged, were grown in glucose at 25°C and then shifted to 37°C for various times as indicated. Cells were treated as described in (B) and the recovered DNA analyzed by QPCR. RSC binding is presented as the percentage immunoprecipitated. (D) RSC binding in the absence of Gal4. Cells deleted for gal4 and bearing TAP-tagged wt RSC were analyzed for RSC binding and MNase sensitivity as described in (C). RSC binding is shown as fold over a control locus in the PHO5 shows that the MNase protection pattern in and around the UASg is not altered by deletion of gal4 . (E) Gal4 and putative RSC binding sites in the UASg . The Gal4 binding sites are indicated in blue. Gal4 sites each bind a dimer of the protein, and each of the three strong binding sites has the sequence CGG-N 11 ) are indicated in red. The rightmost putative RSC site overlaps Gal4 binding site 4, which differs from the Gal4 consensus in one base pair as shown. The arrow indicates the site of truncation of the UASg in the mutant strain created for the experiments in (F). Sequences to the right of the arrow are deleted in the truncation mutant. (F) RSC binding to a wt and a truncated UASg . Cells bearing TAP-tagged RSC and grown in raffinose were probed as described in (C).

    Article Snippet: Nucleosomes bearing H2A.Z at this site were reported by ( ). displays MNase-protected fragments assayed using paired-end sequencing (Illumina).

    Techniques: Binding Assay, Sonication, Isolation, Purification, High Throughput Screening Assay, DNA Sequencing, Mutagenesis, Real-time Polymerase Chain Reaction, Immunoprecipitation, Sequencing

    Relationships between global nucleosome positioning, genomic features, transcription, DNMTs, and DNA methylation ( A ) Normalized distribution profiles for nucleosome binding across intragenic regions for all, protein coding, and non-protein coding genes in pluripotent NCCIT cells. MNase-seq data from human CD4 T cells was used to create similar tag density plots. TSS and TTS (+/− 4kb)-centered plots are also shown to emphasize similarities and differences in these regions. ( B ) Tag density plots for nucleosome distributions across intragenic loci stratified according to promoter CpG density in UD NCCIT (left) and CD4 T cells (right). ( C ) Nucleosome distribution according to expression level in UD (left) and DF NCCIT (right) conditions. ( D ) Representative MNase-seq results at two loci that show among the largest change in expression upon differentiation of NCCIT cells. NANOG is down-regulated and this is accompanied by increased nucleosome density at the TSS. HOXB9 is up-regulated and shows the opposite trend in TSS nucleosome density. The browser data enclosed by blue brackets is enlarged at the right of each panel (red boxed region) with blue ovals indicating inferred positions of nucleosomes. ( E ), nucleosome bound (red) and free (blue) DNA was defined and the correlation between these two states and regions enriched for DNMTs and DNA methylation was calculated. Enrichments are shown as box plots, with the black bar indicating the median, the box defining the first and third quartiles of the data, and whiskers indicating the range (excluding outliers). All bound versus free (linker) comparisons for a given mark are highly significant (P

    Journal: Cell reports

    Article Title: Linking DNA Methyltransferases (DNMTs) to Epigenetic Marks and Nucleosome Structure Genome-Wide in Human Tumor Cells

    doi: 10.1016/j.celrep.2012.10.017

    Figure Lengend Snippet: Relationships between global nucleosome positioning, genomic features, transcription, DNMTs, and DNA methylation ( A ) Normalized distribution profiles for nucleosome binding across intragenic regions for all, protein coding, and non-protein coding genes in pluripotent NCCIT cells. MNase-seq data from human CD4 T cells was used to create similar tag density plots. TSS and TTS (+/− 4kb)-centered plots are also shown to emphasize similarities and differences in these regions. ( B ) Tag density plots for nucleosome distributions across intragenic loci stratified according to promoter CpG density in UD NCCIT (left) and CD4 T cells (right). ( C ) Nucleosome distribution according to expression level in UD (left) and DF NCCIT (right) conditions. ( D ) Representative MNase-seq results at two loci that show among the largest change in expression upon differentiation of NCCIT cells. NANOG is down-regulated and this is accompanied by increased nucleosome density at the TSS. HOXB9 is up-regulated and shows the opposite trend in TSS nucleosome density. The browser data enclosed by blue brackets is enlarged at the right of each panel (red boxed region) with blue ovals indicating inferred positions of nucleosomes. ( E ), nucleosome bound (red) and free (blue) DNA was defined and the correlation between these two states and regions enriched for DNMTs and DNA methylation was calculated. Enrichments are shown as box plots, with the black bar indicating the median, the box defining the first and third quartiles of the data, and whiskers indicating the range (excluding outliers). All bound versus free (linker) comparisons for a given mark are highly significant (P

    Article Snippet: DNA fragments isolated by ChIP, MBD enrichment, or MNase digestion, were used to create single-end sequencing libraries using the Genomic DNA Sample Prep Kit (Illumina) with only minor modifications, as described in the .

    Techniques: DNA Methylation Assay, Binding Assay, Expressing

    Genome-wide nucleosome positioning in Dictyostelium . ( A ) Normalized read midpoint frequency distributions of MNase-protected fragments (nucleosome dyads) of all 12,750 genes in growth-stage WT cells were aligned relative to their ATG codons. Peaks (arrows) correspond to dyad midpoints for globally phased nucleosomes in the 5′ region of intragenic DNA, and distances between mapped read peaks correspond to ∼170 bp NRL. The protein coding DNA sequence (cds) region is shaded. ( B ) Normalized read midpoint frequency distributions of all genes in growth-stage WT cells were aligned relative to their translational termination sites (stop codons). Peaks (arrows) in the mean normalized frequency distribution correspond to globally phased nucleosomes in the 3′ region of intragenic DNA. The protein cds region is shaded. ( C ) Normalized dyad read midpoint frequency distributions for WT chromatin (CHR; dotted line) (see A ) were adjusted for sequence mappability by dividing with equivalent control data from MNase-digested naked (protein free) WT DNA (DNA; red line) and replotted as the ratio (CHR/DNA; thick black line) within 1.2 kb of flanking chromatin relative to ATG sites of all 12,750 genes. An ∼170-bp nucleosome-depleted (“free”) region (NDR) is centered near the AT-rich regions of Dictyostelium TSS. Positioned nucleosomes upstream (+) and downstream (−) to the NDR are indicated by arrows. The protein cds region is shaded.

    Journal: Genome Research

    Article Title: Regulation of nucleosome positioning by a CHD Type III chromatin remodeler and its relationship to developmental gene expression in Dictyostelium

    doi: 10.1101/gr.216309.116

    Figure Lengend Snippet: Genome-wide nucleosome positioning in Dictyostelium . ( A ) Normalized read midpoint frequency distributions of MNase-protected fragments (nucleosome dyads) of all 12,750 genes in growth-stage WT cells were aligned relative to their ATG codons. Peaks (arrows) correspond to dyad midpoints for globally phased nucleosomes in the 5′ region of intragenic DNA, and distances between mapped read peaks correspond to ∼170 bp NRL. The protein coding DNA sequence (cds) region is shaded. ( B ) Normalized read midpoint frequency distributions of all genes in growth-stage WT cells were aligned relative to their translational termination sites (stop codons). Peaks (arrows) in the mean normalized frequency distribution correspond to globally phased nucleosomes in the 3′ region of intragenic DNA. The protein cds region is shaded. ( C ) Normalized dyad read midpoint frequency distributions for WT chromatin (CHR; dotted line) (see A ) were adjusted for sequence mappability by dividing with equivalent control data from MNase-digested naked (protein free) WT DNA (DNA; red line) and replotted as the ratio (CHR/DNA; thick black line) within 1.2 kb of flanking chromatin relative to ATG sites of all 12,750 genes. An ∼170-bp nucleosome-depleted (“free”) region (NDR) is centered near the AT-rich regions of Dictyostelium TSS. Positioned nucleosomes upstream (+) and downstream (−) to the NDR are indicated by arrows. The protein cds region is shaded.

    Article Snippet: Chromatin was digested with micrococcal nuclease (MNase) to create nuclease-resistant DNA ladders with a fragment spectrum of < 1 kb and was analyzed by Illumina paired-end DNA sequencing.

    Techniques: Genome Wide, Sequencing

    ( A ) DNA from MNase digested chromatin fractions purified from wild-type reference yeast strain BY4742 and an isogenic Δcbf1 mutant and separated by agarose gel electrophoresis. The lanes marked: ‘Total’ and ‘Input’ show the DNA species purified from MNase digested cells before and after a brief centrifugation step (to remove high-molecular weight material; see ‘Materials and Methods’ section) respectively. The ‘Input’ fractions were used for sequencing. Marker sizes are shown in kilobases and bands corresponding to DNA from mono-, di- and tri-nucleosomes are indicated to the right of the gel. ( B ) End-to-end distances of paired sequence reads reflect the distribution of chromatin particle input DNA. Graph of the number of Bowtie-aligned ( 9 ) paired-end reads obtained by Illumina GAIIx sequencing of material shown in Figure 1 A ‘Input’ lanes versus paired-read end-to-end distance (Bowtie SAM format insert size value). The values shown on the x -axis indicate aligned sequences with the initial read mapping to the R/+/Crick strand of the reference genome. F/–/Watson strand reads show an identical distribution ( Supplementary Figure S1 ). Peaks at ∼150, 300 and 450 bp are marked and correspond to mono-, di- and tri-nucleosome DNA fractions respectively. ( C ) Paired read size class dyad frequencies describe a landscape of MNase-protected species surrounding protein-coding gene TSSs. Using wild-type cell data, cumulative dyad frequencies for 15-bp bins, centred on and surrounding the protein coding gene TSSs mapped by ( 12 ), were plotted for paired read size classes 36 and 50– 700 bp in 25-bp intervals as a surface graph. Dyad frequencies within each size class were normalized to the mean number of reads per bin for that size class in order to plot each data set on the same y -axis. Normalized cumulative frequency values > 1 are coloured yellow; values

    Journal: Nucleic Acids Research

    Article Title: Chromatin particle spectrum analysis: a method for comparative chromatin structure analysis using paired-end mode next-generation DNA sequencing

    doi: 10.1093/nar/gkq1183

    Figure Lengend Snippet: ( A ) DNA from MNase digested chromatin fractions purified from wild-type reference yeast strain BY4742 and an isogenic Δcbf1 mutant and separated by agarose gel electrophoresis. The lanes marked: ‘Total’ and ‘Input’ show the DNA species purified from MNase digested cells before and after a brief centrifugation step (to remove high-molecular weight material; see ‘Materials and Methods’ section) respectively. The ‘Input’ fractions were used for sequencing. Marker sizes are shown in kilobases and bands corresponding to DNA from mono-, di- and tri-nucleosomes are indicated to the right of the gel. ( B ) End-to-end distances of paired sequence reads reflect the distribution of chromatin particle input DNA. Graph of the number of Bowtie-aligned ( 9 ) paired-end reads obtained by Illumina GAIIx sequencing of material shown in Figure 1 A ‘Input’ lanes versus paired-read end-to-end distance (Bowtie SAM format insert size value). The values shown on the x -axis indicate aligned sequences with the initial read mapping to the R/+/Crick strand of the reference genome. F/–/Watson strand reads show an identical distribution ( Supplementary Figure S1 ). Peaks at ∼150, 300 and 450 bp are marked and correspond to mono-, di- and tri-nucleosome DNA fractions respectively. ( C ) Paired read size class dyad frequencies describe a landscape of MNase-protected species surrounding protein-coding gene TSSs. Using wild-type cell data, cumulative dyad frequencies for 15-bp bins, centred on and surrounding the protein coding gene TSSs mapped by ( 12 ), were plotted for paired read size classes 36 and 50– 700 bp in 25-bp intervals as a surface graph. Dyad frequencies within each size class were normalized to the mean number of reads per bin for that size class in order to plot each data set on the same y -axis. Normalized cumulative frequency values > 1 are coloured yellow; values

    Article Snippet: The MNase-digested genomic ‘Input’ DNA fractions, shown in A, were sequenced in single Illumina GAIIx flowcell lanes to produce 76-bp paired-end reads using standard Illumina protocols and reagents but with the following modifications: DNA nebulization was omitted; adapter ligates were gel-purified over the full size range of input DNA; ligates were hybridized at a low flowcell cluster density.

    Techniques: Purification, Mutagenesis, Agarose Gel Electrophoresis, Centrifugation, Molecular Weight, Sequencing, Marker