Structured Review

Millipore mnase
rRNA genes are switched off in chromatin. ( A ) Reconstitution of mononucleosomes on a 330 bp (–175 to +155) rDNA fragment. The end-labelled DNA was reconstituted into mononucleosomes by salt dialysis reconstitution (lanes 1 and 2) and analysed by native PAGE. <t>Nucleosomal</t> DNA molecules harbouring a nucleosome on the transcription start site were selected by digestion with the restriction enzyme Rsa I (lane 3). The positions of the nucleosomal DNA, the undigested and digested DNA fragment are indicated. ( B ) Transcription assay with free and nucleosomal rDNA fragments. Increasing amounts of free DNA (lanes 1 and 2), a mixture of free DNA and nucleosomal DNA (lanes 3 and 4) and Rsa I-selected nucleosomal DNA (lanes 5–7) were incubated with the transcription extract. The radioactive labelled transcripts were analysed by native PAGE. The nucleosomal templates used for the transcription reactions are shown above the gel. The positions of the undigested or nucleosomal rDNA fragment, the digested free DNA and the 155-nt-long transcript are indicated on the right. ( C ) Analysis of nucleosome positions on the rDNA promoter. Mononucleosomal templates (–175 to +155) were digested with <t>MNase,</t> and the protected nucleosomal DNA was gel-purified, cloned and sequenced. The graph shows the positions of the nucleosomal dyad axis. The positions of the nucAct and nucRep nucleosomes observed in vivo are indicated with the 5′, 3′ and dyad axis positions relative to the rRNA gene transcription start site. E1 and E2 indicate the dyad axis positions of nucleosomes located at the end of the DNA fragment. ( D ) Prediction of nucleosome positioning by the probability of nucleosome occupancy and the probability of encountering a nucleosomal start site. rRNA sequences from position –5000 to +5000 relative to the transcription start site were used for computational analysis at http://genie.weizmann.ac.il/pubs/nucleosomes06/ ( 13 ). The graph displays a window of the calculated predictions, ranging from position –300 to +300 within the rDNA sequence. Peaks of high p (nucleosomal start) values, indicating a high probability for a nucleosomal start site, are indicated. The two nucleosomal positions identified on the rRNA gene in vivo are indicated [ nucAct –157 to –2 (green); the repressive nucleosome position –132 to +22 (red)].
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1) Product Images from "DNA sequence encoded repression of rRNA gene transcription in chromatin"

Article Title: DNA sequence encoded repression of rRNA gene transcription in chromatin

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq263

rRNA genes are switched off in chromatin. ( A ) Reconstitution of mononucleosomes on a 330 bp (–175 to +155) rDNA fragment. The end-labelled DNA was reconstituted into mononucleosomes by salt dialysis reconstitution (lanes 1 and 2) and analysed by native PAGE. Nucleosomal DNA molecules harbouring a nucleosome on the transcription start site were selected by digestion with the restriction enzyme Rsa I (lane 3). The positions of the nucleosomal DNA, the undigested and digested DNA fragment are indicated. ( B ) Transcription assay with free and nucleosomal rDNA fragments. Increasing amounts of free DNA (lanes 1 and 2), a mixture of free DNA and nucleosomal DNA (lanes 3 and 4) and Rsa I-selected nucleosomal DNA (lanes 5–7) were incubated with the transcription extract. The radioactive labelled transcripts were analysed by native PAGE. The nucleosomal templates used for the transcription reactions are shown above the gel. The positions of the undigested or nucleosomal rDNA fragment, the digested free DNA and the 155-nt-long transcript are indicated on the right. ( C ) Analysis of nucleosome positions on the rDNA promoter. Mononucleosomal templates (–175 to +155) were digested with MNase, and the protected nucleosomal DNA was gel-purified, cloned and sequenced. The graph shows the positions of the nucleosomal dyad axis. The positions of the nucAct and nucRep nucleosomes observed in vivo are indicated with the 5′, 3′ and dyad axis positions relative to the rRNA gene transcription start site. E1 and E2 indicate the dyad axis positions of nucleosomes located at the end of the DNA fragment. ( D ) Prediction of nucleosome positioning by the probability of nucleosome occupancy and the probability of encountering a nucleosomal start site. rRNA sequences from position –5000 to +5000 relative to the transcription start site were used for computational analysis at http://genie.weizmann.ac.il/pubs/nucleosomes06/ ( 13 ). The graph displays a window of the calculated predictions, ranging from position –300 to +300 within the rDNA sequence. Peaks of high p (nucleosomal start) values, indicating a high probability for a nucleosomal start site, are indicated. The two nucleosomal positions identified on the rRNA gene in vivo are indicated [ nucAct –157 to –2 (green); the repressive nucleosome position –132 to +22 (red)].
Figure Legend Snippet: rRNA genes are switched off in chromatin. ( A ) Reconstitution of mononucleosomes on a 330 bp (–175 to +155) rDNA fragment. The end-labelled DNA was reconstituted into mononucleosomes by salt dialysis reconstitution (lanes 1 and 2) and analysed by native PAGE. Nucleosomal DNA molecules harbouring a nucleosome on the transcription start site were selected by digestion with the restriction enzyme Rsa I (lane 3). The positions of the nucleosomal DNA, the undigested and digested DNA fragment are indicated. ( B ) Transcription assay with free and nucleosomal rDNA fragments. Increasing amounts of free DNA (lanes 1 and 2), a mixture of free DNA and nucleosomal DNA (lanes 3 and 4) and Rsa I-selected nucleosomal DNA (lanes 5–7) were incubated with the transcription extract. The radioactive labelled transcripts were analysed by native PAGE. The nucleosomal templates used for the transcription reactions are shown above the gel. The positions of the undigested or nucleosomal rDNA fragment, the digested free DNA and the 155-nt-long transcript are indicated on the right. ( C ) Analysis of nucleosome positions on the rDNA promoter. Mononucleosomal templates (–175 to +155) were digested with MNase, and the protected nucleosomal DNA was gel-purified, cloned and sequenced. The graph shows the positions of the nucleosomal dyad axis. The positions of the nucAct and nucRep nucleosomes observed in vivo are indicated with the 5′, 3′ and dyad axis positions relative to the rRNA gene transcription start site. E1 and E2 indicate the dyad axis positions of nucleosomes located at the end of the DNA fragment. ( D ) Prediction of nucleosome positioning by the probability of nucleosome occupancy and the probability of encountering a nucleosomal start site. rRNA sequences from position –5000 to +5000 relative to the transcription start site were used for computational analysis at http://genie.weizmann.ac.il/pubs/nucleosomes06/ ( 13 ). The graph displays a window of the calculated predictions, ranging from position –300 to +300 within the rDNA sequence. Peaks of high p (nucleosomal start) values, indicating a high probability for a nucleosomal start site, are indicated. The two nucleosomal positions identified on the rRNA gene in vivo are indicated [ nucAct –157 to –2 (green); the repressive nucleosome position –132 to +22 (red)].

Techniques Used: Clear Native PAGE, Incubation, Purification, Clone Assay, In Vivo, Sequencing

PK inhibits nucleosome remodelling at the rDNA promoter. ( A ) MNase digestion of chromatin assembled on the rDNA minigene (pMrWT-T). Chromatin reconstituted with the Drosophila extract was digested with MNase for 0.5–3 min (lanes 1–3) or for 0.5–6 min (lanes 4–8) in the presence of 600 μM PK. The nucleosomal ladder (1n-5n) and the DNA marker (M; 1kb ladder) are indicated. ( B ) Chromatin assembled on pMrWT-T was incubated in the absence or presence of TTF-I and partially digested with MNase. Purified DNA was digested with EcoRI, separated on an agarose gel and transferred onto a nylon membrane. Chromatin configuration around the TTF-I-binding site (T 0 ) was visualized by indirect end-labelling (lanes 1 and 2). Chromatin remodelling was monitored in the presence of K (lanes 4 and 5; 300 and 600 μM) or PK (lanes 6 and 7, 300 and 600 μM). The position of the TTF-I-binding site is indicated by Sal I digestion of the template DNA (lane 3). Open circles mark non-positioned nucleosomes, whereas the gray circles indicate positioned nucleosomes. The position of the TTF-I-binding site (gray box), MNase-protected DNA regions (black triangles) and MNase-sensitive regions (white triangles) are indicated. The strong band in lane 6 (marked with an asterisk) arises due to the relatively lower MNase digestion of this sample.
Figure Legend Snippet: PK inhibits nucleosome remodelling at the rDNA promoter. ( A ) MNase digestion of chromatin assembled on the rDNA minigene (pMrWT-T). Chromatin reconstituted with the Drosophila extract was digested with MNase for 0.5–3 min (lanes 1–3) or for 0.5–6 min (lanes 4–8) in the presence of 600 μM PK. The nucleosomal ladder (1n-5n) and the DNA marker (M; 1kb ladder) are indicated. ( B ) Chromatin assembled on pMrWT-T was incubated in the absence or presence of TTF-I and partially digested with MNase. Purified DNA was digested with EcoRI, separated on an agarose gel and transferred onto a nylon membrane. Chromatin configuration around the TTF-I-binding site (T 0 ) was visualized by indirect end-labelling (lanes 1 and 2). Chromatin remodelling was monitored in the presence of K (lanes 4 and 5; 300 and 600 μM) or PK (lanes 6 and 7, 300 and 600 μM). The position of the TTF-I-binding site is indicated by Sal I digestion of the template DNA (lane 3). Open circles mark non-positioned nucleosomes, whereas the gray circles indicate positioned nucleosomes. The position of the TTF-I-binding site (gray box), MNase-protected DNA regions (black triangles) and MNase-sensitive regions (white triangles) are indicated. The strong band in lane 6 (marked with an asterisk) arises due to the relatively lower MNase digestion of this sample.

Techniques Used: Marker, Incubation, Purification, Agarose Gel Electrophoresis, Binding Assay

TTF-I-dependent chromatin dynamics at the rRNA gene promoter. Reconstituted nucleosomal arrays were incubated for 90 min with the TxE, TTF-I, K (600 μM) or PK (600 μM) as indicated. Nucleosome positions at the rDNA promoter were mapped by partial MNase digestion and primer extension of the purified DNA. DNA fragments were resolved on 8% sequencing gels and quantified with a PhosphorImager. The graph shows the positions (relative to the transcription start site, +1; site is marked by boxes) and relative intensities of the MNase cleavage sites corresponding to the 3′ boundaries of positioned nucleosomes. Boxes highlight the MNase cleavage sites around the transcription start site, correlating with the nucleosome position nucAct . The position of the oligonucleotide used for primer extension and the major MNase-sensitive sites on the rDNA are indicated. The scan of the DNA marker (10-bp ladder) is shown below the graphs.
Figure Legend Snippet: TTF-I-dependent chromatin dynamics at the rRNA gene promoter. Reconstituted nucleosomal arrays were incubated for 90 min with the TxE, TTF-I, K (600 μM) or PK (600 μM) as indicated. Nucleosome positions at the rDNA promoter were mapped by partial MNase digestion and primer extension of the purified DNA. DNA fragments were resolved on 8% sequencing gels and quantified with a PhosphorImager. The graph shows the positions (relative to the transcription start site, +1; site is marked by boxes) and relative intensities of the MNase cleavage sites corresponding to the 3′ boundaries of positioned nucleosomes. Boxes highlight the MNase cleavage sites around the transcription start site, correlating with the nucleosome position nucAct . The position of the oligonucleotide used for primer extension and the major MNase-sensitive sites on the rDNA are indicated. The scan of the DNA marker (10-bp ladder) is shown below the graphs.

Techniques Used: Incubation, Purification, Sequencing, Marker

2) Product Images from "Distinct roles for S. cerevisiae H2A copies in recombination and repeat stability, with a role for H2A.1 threonine 126"

Article Title: Distinct roles for S. cerevisiae H2A copies in recombination and repeat stability, with a role for H2A.1 threonine 126

Journal: eLife

doi: 10.7554/eLife.53362

Nucleosome positioning at a (CAG) 85 repeat is not altered in the absence of H2A.1 or H2A.2. A ) Indirect end-labeling of nucleosomal DNA upstream of the CAG repeat. MNase (0, 0.25, 2.5, and 7.5 units) digested DNA was run in 1.5% agarose with ethidium bromide (left) and Southern blotted (right) using a probe ~100 bp proximal to the CAG repeat (red line Figure 1—figure supplement 1A ). Ovals represent nucleosome positions. The experiment was repeated six times; a representative blot is shown. ( B ) Illumina array mapping of nucleosome protection at the CAG repeat. Mononucleosomal DNA from strains containing the (CAG) 85 repeats was hybridized to a custom array of 30-mer probes spanning 425 bp upstream of the repeat to 436 bp downstream of the repeat in YAC CF1. Probes 14–16 contain CAG repeats; probe 15 is composed purely of CAG repeats (probe sequences in Supplementary file 3 ). Error bars represent standard deviation of 2–3 independent experiments.
Figure Legend Snippet: Nucleosome positioning at a (CAG) 85 repeat is not altered in the absence of H2A.1 or H2A.2. A ) Indirect end-labeling of nucleosomal DNA upstream of the CAG repeat. MNase (0, 0.25, 2.5, and 7.5 units) digested DNA was run in 1.5% agarose with ethidium bromide (left) and Southern blotted (right) using a probe ~100 bp proximal to the CAG repeat (red line Figure 1—figure supplement 1A ). Ovals represent nucleosome positions. The experiment was repeated six times; a representative blot is shown. ( B ) Illumina array mapping of nucleosome protection at the CAG repeat. Mononucleosomal DNA from strains containing the (CAG) 85 repeats was hybridized to a custom array of 30-mer probes spanning 425 bp upstream of the repeat to 436 bp downstream of the repeat in YAC CF1. Probes 14–16 contain CAG repeats; probe 15 is composed purely of CAG repeats (probe sequences in Supplementary file 3 ). Error bars represent standard deviation of 2–3 independent experiments.

Techniques Used: End Labeling, Standard Deviation

Nucleosome positioning at a (CAG) 85 repeat is not altered in the absence of H2A.1 or H2A.2. A ) Indirect end-labeling of nucleosomal DNA upstream of the CAG repeat. MNase (0, 0.25, 2.5, and 7.5 units) digested DNA was run in 1.5% agarose with ethidium bromide (left) and Southern blotted (right) using a probe ~100 bp proximal to the CAG repeat (red line Figure 1—figure supplement 1A ). Ovals represent nucleosome positions. The experiment was repeated six times; a representative blot is shown. ( B ) Illumina array mapping of nucleosome protection at the CAG repeat. Mononucleosomal DNA from strains containing the (CAG) 85 repeats was hybridized to a custom array of 30-mer probes spanning 425 bp upstream of the repeat to 436 bp downstream of the repeat in YAC CF1. Probes 14–16 contain CAG repeats; probe 15 is composed purely of CAG repeats (probe sequences in Supplementary file 3 ). Error bars represent standard deviation of 2–3 independent experiments.
Figure Legend Snippet: Nucleosome positioning at a (CAG) 85 repeat is not altered in the absence of H2A.1 or H2A.2. A ) Indirect end-labeling of nucleosomal DNA upstream of the CAG repeat. MNase (0, 0.25, 2.5, and 7.5 units) digested DNA was run in 1.5% agarose with ethidium bromide (left) and Southern blotted (right) using a probe ~100 bp proximal to the CAG repeat (red line Figure 1—figure supplement 1A ). Ovals represent nucleosome positions. The experiment was repeated six times; a representative blot is shown. ( B ) Illumina array mapping of nucleosome protection at the CAG repeat. Mononucleosomal DNA from strains containing the (CAG) 85 repeats was hybridized to a custom array of 30-mer probes spanning 425 bp upstream of the repeat to 436 bp downstream of the repeat in YAC CF1. Probes 14–16 contain CAG repeats; probe 15 is composed purely of CAG repeats (probe sequences in Supplementary file 3 ). Error bars represent standard deviation of 2–3 independent experiments.

Techniques Used: End Labeling, Standard Deviation

3) Product Images from "Distinct roles for S. cerevisiae H2A copies in recombination and repeat stability, with a role for H2A.1 threonine 126"

Article Title: Distinct roles for S. cerevisiae H2A copies in recombination and repeat stability, with a role for H2A.1 threonine 126

Journal: eLife

doi: 10.7554/eLife.53362

Nucleosome positioning at a (CAG) 85 repeat is not altered in the absence of H2A.1 or H2A.2. A ) Indirect end-labeling of nucleosomal DNA upstream of the CAG repeat. MNase (0, 0.25, 2.5, and 7.5 units) digested DNA was run in 1.5% agarose with ethidium bromide (left) and Southern blotted (right) using a probe ~100 bp proximal to the CAG repeat (red line Figure 1—figure supplement 1A ). Ovals represent nucleosome positions. The experiment was repeated six times; a representative blot is shown. ( B ) Illumina array mapping of nucleosome protection at the CAG repeat. Mononucleosomal DNA from strains containing the (CAG) 85 repeats was hybridized to a custom array of 30-mer probes spanning 425 bp upstream of the repeat to 436 bp downstream of the repeat in YAC CF1. Probes 14–16 contain CAG repeats; probe 15 is composed purely of CAG repeats (probe sequences in Supplementary file 3 ). Error bars represent standard deviation of 2–3 independent experiments.
Figure Legend Snippet: Nucleosome positioning at a (CAG) 85 repeat is not altered in the absence of H2A.1 or H2A.2. A ) Indirect end-labeling of nucleosomal DNA upstream of the CAG repeat. MNase (0, 0.25, 2.5, and 7.5 units) digested DNA was run in 1.5% agarose with ethidium bromide (left) and Southern blotted (right) using a probe ~100 bp proximal to the CAG repeat (red line Figure 1—figure supplement 1A ). Ovals represent nucleosome positions. The experiment was repeated six times; a representative blot is shown. ( B ) Illumina array mapping of nucleosome protection at the CAG repeat. Mononucleosomal DNA from strains containing the (CAG) 85 repeats was hybridized to a custom array of 30-mer probes spanning 425 bp upstream of the repeat to 436 bp downstream of the repeat in YAC CF1. Probes 14–16 contain CAG repeats; probe 15 is composed purely of CAG repeats (probe sequences in Supplementary file 3 ). Error bars represent standard deviation of 2–3 independent experiments.

Techniques Used: End Labeling, Standard Deviation

Nucleosome positioning at a (CAG) 85 repeat is not altered in the absence of H2A.1 or H2A.2. A ) Indirect end-labeling of nucleosomal DNA upstream of the CAG repeat. MNase (0, 0.25, 2.5, and 7.5 units) digested DNA was run in 1.5% agarose with ethidium bromide (left) and Southern blotted (right) using a probe ~100 bp proximal to the CAG repeat (red line Figure 1—figure supplement 1A ). Ovals represent nucleosome positions. The experiment was repeated six times; a representative blot is shown. ( B ) Illumina array mapping of nucleosome protection at the CAG repeat. Mononucleosomal DNA from strains containing the (CAG) 85 repeats was hybridized to a custom array of 30-mer probes spanning 425 bp upstream of the repeat to 436 bp downstream of the repeat in YAC CF1. Probes 14–16 contain CAG repeats; probe 15 is composed purely of CAG repeats (probe sequences in Supplementary file 3 ). Error bars represent standard deviation of 2–3 independent experiments.
Figure Legend Snippet: Nucleosome positioning at a (CAG) 85 repeat is not altered in the absence of H2A.1 or H2A.2. A ) Indirect end-labeling of nucleosomal DNA upstream of the CAG repeat. MNase (0, 0.25, 2.5, and 7.5 units) digested DNA was run in 1.5% agarose with ethidium bromide (left) and Southern blotted (right) using a probe ~100 bp proximal to the CAG repeat (red line Figure 1—figure supplement 1A ). Ovals represent nucleosome positions. The experiment was repeated six times; a representative blot is shown. ( B ) Illumina array mapping of nucleosome protection at the CAG repeat. Mononucleosomal DNA from strains containing the (CAG) 85 repeats was hybridized to a custom array of 30-mer probes spanning 425 bp upstream of the repeat to 436 bp downstream of the repeat in YAC CF1. Probes 14–16 contain CAG repeats; probe 15 is composed purely of CAG repeats (probe sequences in Supplementary file 3 ). Error bars represent standard deviation of 2–3 independent experiments.

Techniques Used: End Labeling, Standard Deviation

4) Product Images from "Synergy between histone deacetylase inhibitors and DNA-damaging agents is mediated by histone deacetylase 2 in colorectal cancer"

Article Title: Synergy between histone deacetylase inhibitors and DNA-damaging agents is mediated by histone deacetylase 2 in colorectal cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.9887

HDAC2 controls the chromatin plasticity and its depletion enhances mitotic cell death in drug resistant HT-29 cells upon 5-FU and Oxa treatments A. PARPc measurement in HT-29 treated by 5-FU and Oxa combined with SAHA. After 24 hours, cells were lysed and the proteins separated using SDS-PAGE. B and C. HT-29 cell lines were treated by 5-FU or Oxa alone or combined with SAHA. After 24 hours, Cells were fixed 4% paraformaldehyde, subsequently DNA was stained with DAPI (0.1 μg/ml; Sigma-Aldrich) and the number of apoptotic cells was measured quantitatively by assessing the percentage of cells with fragmented or condensed nuclei. Mitotic cell death (MCD) was quantified by using phosphorylated histone 3 (ser10) as a mitotic cell marker. C) Representative image of mitotic cell death (MCD) in HT-29 upon SAHA + Oxa combined treatment. D. HT-29 or shRNA-HDAC2 HT-29 cells lines were treated with 5-FU or Oxa only or in combination with SAHA. After 24 hours, cells were lysed and the proteins separated using SDS-PAGE. The PARPc and the protein level of HDAC2 were analyzed by WB. Actin was used as a loading control. E. HT-29 cell lines or shRNA-HDAC2 HT-29 cells lines were treated by 5-FU, Oxa or SAHA. After 24 hours, mitotic cell death (MCD) was quantified by using phosphorylated histone 3 (ser10) as a mitotic cell marker. F. HT-29 cell lines were treated by Oxa alone or combined with SAHA and shRNA-HDAC2 HT-29 cells were treated with Oxa. After 24 hours, cells were fixed and HDAC2 protein was detected after immunofluorescence staining. Nucleus was counterstained using DAPI staining. + z-Stack shows nuclear deformation. G. MNase accessibility assay was used to study relaxed chromatin which has higher accessibility to micrococal nuclease enzyme (MNase). Cells HT-29 cells were treated with Oxa or SAHA alone or combined for 24hr and chromatin was extracted and incubated with 0.06U of MNase and fragmented DNA was separated by gel agarose, the arrow represent the undigested DNA. For all the experiments error bars represent ± S.E.M. of three independent experiments ( n =3) and statistical significance is depicted by * for p
Figure Legend Snippet: HDAC2 controls the chromatin plasticity and its depletion enhances mitotic cell death in drug resistant HT-29 cells upon 5-FU and Oxa treatments A. PARPc measurement in HT-29 treated by 5-FU and Oxa combined with SAHA. After 24 hours, cells were lysed and the proteins separated using SDS-PAGE. B and C. HT-29 cell lines were treated by 5-FU or Oxa alone or combined with SAHA. After 24 hours, Cells were fixed 4% paraformaldehyde, subsequently DNA was stained with DAPI (0.1 μg/ml; Sigma-Aldrich) and the number of apoptotic cells was measured quantitatively by assessing the percentage of cells with fragmented or condensed nuclei. Mitotic cell death (MCD) was quantified by using phosphorylated histone 3 (ser10) as a mitotic cell marker. C) Representative image of mitotic cell death (MCD) in HT-29 upon SAHA + Oxa combined treatment. D. HT-29 or shRNA-HDAC2 HT-29 cells lines were treated with 5-FU or Oxa only or in combination with SAHA. After 24 hours, cells were lysed and the proteins separated using SDS-PAGE. The PARPc and the protein level of HDAC2 were analyzed by WB. Actin was used as a loading control. E. HT-29 cell lines or shRNA-HDAC2 HT-29 cells lines were treated by 5-FU, Oxa or SAHA. After 24 hours, mitotic cell death (MCD) was quantified by using phosphorylated histone 3 (ser10) as a mitotic cell marker. F. HT-29 cell lines were treated by Oxa alone or combined with SAHA and shRNA-HDAC2 HT-29 cells were treated with Oxa. After 24 hours, cells were fixed and HDAC2 protein was detected after immunofluorescence staining. Nucleus was counterstained using DAPI staining. + z-Stack shows nuclear deformation. G. MNase accessibility assay was used to study relaxed chromatin which has higher accessibility to micrococal nuclease enzyme (MNase). Cells HT-29 cells were treated with Oxa or SAHA alone or combined for 24hr and chromatin was extracted and incubated with 0.06U of MNase and fragmented DNA was separated by gel agarose, the arrow represent the undigested DNA. For all the experiments error bars represent ± S.E.M. of three independent experiments ( n =3) and statistical significance is depicted by * for p

Techniques Used: SDS Page, Staining, Marker, shRNA, Western Blot, Immunofluorescence, Incubation

5) Product Images from "Histone octamer trans-transfer: a signature mechanism of ATP-dependent chromatin remodelling unravelled in wheat nuclear extract"

Article Title: Histone octamer trans-transfer: a signature mechanism of ATP-dependent chromatin remodelling unravelled in wheat nuclear extract

Journal: Annals of Botany

doi: 10.1093/aob/mcr232

Profile of DNA isolated from the soluble chromatin: DNA was extracted from fractionated nucleosomes obtained from chromatin derived from MNase-treated wheat nuclei and analysed on 1 % agarose gel. The lane numbers (25–39) indicate fraction numbers.
Figure Legend Snippet: Profile of DNA isolated from the soluble chromatin: DNA was extracted from fractionated nucleosomes obtained from chromatin derived from MNase-treated wheat nuclei and analysed on 1 % agarose gel. The lane numbers (25–39) indicate fraction numbers.

Techniques Used: Isolation, Derivative Assay, Agarose Gel Electrophoresis

6) Product Images from "DNA sequence- and conformation-directed positioning of nucleosomes by chromatin-remodeling complexes"

Article Title: DNA sequence- and conformation-directed positioning of nucleosomes by chromatin-remodeling complexes

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0702430104

A short DNA element can direct ACF-dependent nucleosome positioning. ( A ) Remodeling reaction with ACF or ISWI with a nucleosome substrate containing a 253-bp-long DNA fragment (K3 DNA) from the pT-K3 plasmid. After nucleosome assembly by salt dialysis on the K3 DNA, a mixed population of a single nucleosome with three main positions (N1, N2, and N4) and one minor position (N3, lane 1) was obtained. This substrate was used in a remodeling reaction with ISWI (lane 2) or ACF (lane 3). ( B ) High-resolution mapping of the remodeler-dependent nucleosome positions on the K3 DNA substrate. MNase protection and subsequent primer extension reactions were conducted. Scans for the primer extension reactions ( Left , forward primer; Right , reverse primer) are shown for the nucleosomal input substrate (green, corresponding to A , lane 1) and the remodeling reaction for ACF (red, corresponding to A , lane 3). The black curve shows a 10-bp DNA marker. The same analysis was conducted with ISWI (data not shown). The peaks reflect nucleosomes positioned adjacent to this site. Considering that 147 bp of DNA are protected by the nucleosome, the major nucleosome positions were identified as 37/45 to 187/195 for N1, 25 to 175 for N2, and 0/7 to 151/157 for N4. ( C ) The ACF- and ISWI-dependent nucleosome positions determined on the 253-bp K3 DNA fragment were plotted together with the predicted DNA curvature. The black arrow refers to the 40-bp DNA sequence encompassing the region of maximal DNA curvature from the rDNA sequence that was cloned into the K3 DNA. ( D ) Same analysis as in C ).
Figure Legend Snippet: A short DNA element can direct ACF-dependent nucleosome positioning. ( A ) Remodeling reaction with ACF or ISWI with a nucleosome substrate containing a 253-bp-long DNA fragment (K3 DNA) from the pT-K3 plasmid. After nucleosome assembly by salt dialysis on the K3 DNA, a mixed population of a single nucleosome with three main positions (N1, N2, and N4) and one minor position (N3, lane 1) was obtained. This substrate was used in a remodeling reaction with ISWI (lane 2) or ACF (lane 3). ( B ) High-resolution mapping of the remodeler-dependent nucleosome positions on the K3 DNA substrate. MNase protection and subsequent primer extension reactions were conducted. Scans for the primer extension reactions ( Left , forward primer; Right , reverse primer) are shown for the nucleosomal input substrate (green, corresponding to A , lane 1) and the remodeling reaction for ACF (red, corresponding to A , lane 3). The black curve shows a 10-bp DNA marker. The same analysis was conducted with ISWI (data not shown). The peaks reflect nucleosomes positioned adjacent to this site. Considering that 147 bp of DNA are protected by the nucleosome, the major nucleosome positions were identified as 37/45 to 187/195 for N1, 25 to 175 for N2, and 0/7 to 151/157 for N4. ( C ) The ACF- and ISWI-dependent nucleosome positions determined on the 253-bp K3 DNA fragment were plotted together with the predicted DNA curvature. The black arrow refers to the 40-bp DNA sequence encompassing the region of maximal DNA curvature from the rDNA sequence that was cloned into the K3 DNA. ( D ) Same analysis as in C ).

Techniques Used: Plasmid Preparation, Marker, Sequencing, Clone Assay

7) Product Images from "Chromatin assembly factor I and Hir proteins contribute to building functional kinetochores in S. cerevisiae"

Article Title: Chromatin assembly factor I and Hir proteins contribute to building functional kinetochores in S. cerevisiae

Journal: Genes & Development

doi: 10.1101/gad.925302

Centromeric chromatin phenotypes in cac1Δ hir1Δ cells. Nuclei were prepared from yeast strains PKY346 (wt), PKY1100 ( cac1Δ ), PKY1154 ( hir1Δ ), and PKY1168 ( cac1Δ hir1Δ ) as indicated and digested with nucleases as follows. ( A – C ) Indirect end-label analysis of CEN3 chromatin. Nuclei were incubated with 0.6 U MNase (Sigma) at 32°C for 0 min (lanes 1,5,9,13 ), 5 min (lanes 2,6,10,14 ), 10 min (lanes 3,7,11,15 ), or 15 min (lanes 4,8,12,16 ) prior to isolation of genomic DNA, restriction enzyme digestion, and DNA blot hybridization with probes as described in the Materials and Methods. ( A ) CDEIII-proximal side of CEN3 in cells grown at 30°C. Samples were digested with Cla I. ( B ) CDEIII-proximal side of CEN3 in cells shifted to 16°C for 36 h prior to isolation of nuclei. Samples were digested with Cla I. ( C ) CDEI-proximal side of CEN3 in nuclei prepared from cells grown at 16°C. Samples were digested with Bam HI. ( D ) Dra I accessibility to CDEII within CEN3 . Nuclei were incubated with Dra I (0, 50, 100, or 150 U/mL) at 37°C for 30 min. DNA was purified, digested with Eco RI, and subjected to Southern blot hybridization. The fold differences in digestion relative to wild-type cells represent the average of three independent experiments with standard deviations indicated by the error bars. ( Lower panel) A region of the same gel used for the Southern blot visualized by ethidium bromide staining, indicating similar extents of Dra I digestion of total chromatin in all four strains.
Figure Legend Snippet: Centromeric chromatin phenotypes in cac1Δ hir1Δ cells. Nuclei were prepared from yeast strains PKY346 (wt), PKY1100 ( cac1Δ ), PKY1154 ( hir1Δ ), and PKY1168 ( cac1Δ hir1Δ ) as indicated and digested with nucleases as follows. ( A – C ) Indirect end-label analysis of CEN3 chromatin. Nuclei were incubated with 0.6 U MNase (Sigma) at 32°C for 0 min (lanes 1,5,9,13 ), 5 min (lanes 2,6,10,14 ), 10 min (lanes 3,7,11,15 ), or 15 min (lanes 4,8,12,16 ) prior to isolation of genomic DNA, restriction enzyme digestion, and DNA blot hybridization with probes as described in the Materials and Methods. ( A ) CDEIII-proximal side of CEN3 in cells grown at 30°C. Samples were digested with Cla I. ( B ) CDEIII-proximal side of CEN3 in cells shifted to 16°C for 36 h prior to isolation of nuclei. Samples were digested with Cla I. ( C ) CDEI-proximal side of CEN3 in nuclei prepared from cells grown at 16°C. Samples were digested with Bam HI. ( D ) Dra I accessibility to CDEII within CEN3 . Nuclei were incubated with Dra I (0, 50, 100, or 150 U/mL) at 37°C for 30 min. DNA was purified, digested with Eco RI, and subjected to Southern blot hybridization. The fold differences in digestion relative to wild-type cells represent the average of three independent experiments with standard deviations indicated by the error bars. ( Lower panel) A region of the same gel used for the Southern blot visualized by ethidium bromide staining, indicating similar extents of Dra I digestion of total chromatin in all four strains.

Techniques Used: Incubation, Isolation, Hybridization, Purification, Southern Blot, Staining

8) Product Images from "Recruitment of the Nucleolar Remodeling Complex NoRC Establishes Ribosomal DNA Silencing in Chromatin"

Article Title: Recruitment of the Nucleolar Remodeling Complex NoRC Establishes Ribosomal DNA Silencing in Chromatin

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.4.1791-1798.2004

NoRC silences rDNA transcription for chromatin templates. (A) Supercoiling assay. Nucleosomes were assembled on pMrWT-T by salt dialysis and purified in a sucrose gradient. Individual fractions were incubated with topoisomerase I, and the topoisomer distribution of the purified DNA was visualized on agarose gels containing chloroquine. Supercoiled DNA (sc, lane 1), partially relaxed DNA (lane 2), and fractions with decreasing nucleosome density (lanes 3 to 8) are shown. The nucleosomal fraction used for the experiments is indicated by a white triangle. (B) MNase digestion. The indicated chromatin fraction was digested with increasing amounts of MNase. Purified DNA was visualized by agarose gel electrophoresis and ethidium bromide staining. The regular fragment ladder indicative of the nucleosomal array is indicated (1n through 6n). (C) Transcription assay. A minigene (pMrWT-T) containing the rDNA promoter and the termination region was used for in vitro transcription. DNA and chromatin were incubated with the transcription extract in the absence or presence of TTF-I (lanes 1 and 2). Readthrough transcription in the absence and terminated transcription in the presence of TTF is indicated on the left. Increasing amounts of Snf2H (lanes 2 to 4; 25, 50, and 100 fmol, respectively), NoRC (lanes 6 to 8; 25, 50, and 100 fmol, respectively) and ACF (9 to 11; 25, 50, and 100 fmol, respectively), were incubated with TTF-I, resulting in terminated transcripts. Transcription was performed for naked DNA and for chromatin templates as indicated.
Figure Legend Snippet: NoRC silences rDNA transcription for chromatin templates. (A) Supercoiling assay. Nucleosomes were assembled on pMrWT-T by salt dialysis and purified in a sucrose gradient. Individual fractions were incubated with topoisomerase I, and the topoisomer distribution of the purified DNA was visualized on agarose gels containing chloroquine. Supercoiled DNA (sc, lane 1), partially relaxed DNA (lane 2), and fractions with decreasing nucleosome density (lanes 3 to 8) are shown. The nucleosomal fraction used for the experiments is indicated by a white triangle. (B) MNase digestion. The indicated chromatin fraction was digested with increasing amounts of MNase. Purified DNA was visualized by agarose gel electrophoresis and ethidium bromide staining. The regular fragment ladder indicative of the nucleosomal array is indicated (1n through 6n). (C) Transcription assay. A minigene (pMrWT-T) containing the rDNA promoter and the termination region was used for in vitro transcription. DNA and chromatin were incubated with the transcription extract in the absence or presence of TTF-I (lanes 1 and 2). Readthrough transcription in the absence and terminated transcription in the presence of TTF is indicated on the left. Increasing amounts of Snf2H (lanes 2 to 4; 25, 50, and 100 fmol, respectively), NoRC (lanes 6 to 8; 25, 50, and 100 fmol, respectively) and ACF (9 to 11; 25, 50, and 100 fmol, respectively), were incubated with TTF-I, resulting in terminated transcripts. Transcription was performed for naked DNA and for chromatin templates as indicated.

Techniques Used: Purification, Incubation, Agarose Gel Electrophoresis, Staining, In Vitro

9) Product Images from "Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y"

Article Title: Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201002043

Structure and stability of H3.X- and H3.Y-containing nucleosomes. (A) In silico homology model of H3.X (purple, left) and H3.Y (light blue, right) protein structures in overlay with the crystal structure of H3.2 (dark blue). (B) Crystal structure of nucleosome with H3.2 exchanged by in silico homology models of H3.X (purple, left) and H3.Y (light blue, right), respectively. (C) IP of mononucleosomes generated from HeLa cells transfected with empty vector, HA-H3.1, -H3.X, and -H3.Y shows incorporation of novel H3 variants into nucleosomes. Bioanalyzer evaluation of purified DNA after IP of MNase-treated chromatin (unbound and bound material) shows digestion of chromatin to mononucleosomes and their successful precipitation (left; see also Fig. S2 A for DNA size and quality). Silver stain of 15% SDS-PAGE with α-HA IPs of mononucleosomes revealed successful binding of HA-tagged H3 variants (asterisks) and pull-down of core histones (top, right). Immunoblot of immunoprecipitates with α-HA (red) and α-H3 C-terminal (green) antibodies visualized by the Odyssey infrared imaging system (bottom, right). Notice that endogenous H3 is coimmunoprecipitated with all H3 variants analyzed. (D) FRAP experiments to evaluate nucleosomal stability of novel H3 variants using spinning disk confocal microscopy. HeLa Kyoto cells were transiently transfected with GFP, GFP-H3.1, -H3.3, -H3.X, and -H3.Y constructs. A small nuclear area was photobleached (box) and the recovery of the fluorescent signal was monitored over 1 min and up to 8 h (see Fig. S2, B–D, for long-term FRAP). Depicted is a short-term FRAP series (selected time points are shown) of GFP-tagged H3 variants compared with GFP alone. Bar, 5 µm. (E) Quantification of short-term FRAP experiment. Mean curves of 10–20 individual cells are shown. Standard deviations were very small (in the range of ± 0.02) and were omitted for clarity (for details see Fig. S2 D). All GFP-H3 variants show almost no recovery within the first 60 s after bleaching, which indicates that all expressed fusion protein was stably incorporated into nucleosomes. In contrast, GFP alone recovers to almost 100% within 5 s.
Figure Legend Snippet: Structure and stability of H3.X- and H3.Y-containing nucleosomes. (A) In silico homology model of H3.X (purple, left) and H3.Y (light blue, right) protein structures in overlay with the crystal structure of H3.2 (dark blue). (B) Crystal structure of nucleosome with H3.2 exchanged by in silico homology models of H3.X (purple, left) and H3.Y (light blue, right), respectively. (C) IP of mononucleosomes generated from HeLa cells transfected with empty vector, HA-H3.1, -H3.X, and -H3.Y shows incorporation of novel H3 variants into nucleosomes. Bioanalyzer evaluation of purified DNA after IP of MNase-treated chromatin (unbound and bound material) shows digestion of chromatin to mononucleosomes and their successful precipitation (left; see also Fig. S2 A for DNA size and quality). Silver stain of 15% SDS-PAGE with α-HA IPs of mononucleosomes revealed successful binding of HA-tagged H3 variants (asterisks) and pull-down of core histones (top, right). Immunoblot of immunoprecipitates with α-HA (red) and α-H3 C-terminal (green) antibodies visualized by the Odyssey infrared imaging system (bottom, right). Notice that endogenous H3 is coimmunoprecipitated with all H3 variants analyzed. (D) FRAP experiments to evaluate nucleosomal stability of novel H3 variants using spinning disk confocal microscopy. HeLa Kyoto cells were transiently transfected with GFP, GFP-H3.1, -H3.3, -H3.X, and -H3.Y constructs. A small nuclear area was photobleached (box) and the recovery of the fluorescent signal was monitored over 1 min and up to 8 h (see Fig. S2, B–D, for long-term FRAP). Depicted is a short-term FRAP series (selected time points are shown) of GFP-tagged H3 variants compared with GFP alone. Bar, 5 µm. (E) Quantification of short-term FRAP experiment. Mean curves of 10–20 individual cells are shown. Standard deviations were very small (in the range of ± 0.02) and were omitted for clarity (for details see Fig. S2 D). All GFP-H3 variants show almost no recovery within the first 60 s after bleaching, which indicates that all expressed fusion protein was stably incorporated into nucleosomes. In contrast, GFP alone recovers to almost 100% within 5 s.

Techniques Used: In Silico, Generated, Transfection, Plasmid Preparation, Purification, Silver Staining, SDS Page, Binding Assay, Imaging, Confocal Microscopy, Construct, Stable Transfection

10) Product Images from "NF-Y Associates with H3-H4 Tetramers and Octamers by Multiple Mechanisms"

Article Title: NF-Y Associates with H3-H4 Tetramers and Octamers by Multiple Mechanisms

Journal: Molecular and Cellular Biology

doi:

MNase accessibility assay of histone–NF-YB–NF-YC combinations. Stoichiometric amounts of the indicated combinations of HFM proteins were reconstituted with fragment 2 (labeled on the top strand [lanes 4 to 9] and bottom strand [lanes 13 to 18]), cut with MNase, and analyzed on sequencing gels. In lane 17, the NF-Y trimer was used to show the NF-Y footprinted area. F refers to free, mock-reconstituted DNA (lanes 4 and 5; uncut and cut with MNase, respectively). Arrows correspond to the major and minor hypersensitive sites. Part of the H3-H4 and H3–H4–NF-YB–NF-YC reconstitutions were cut with DNase I and run in parallel (lanes 1, 2, 10, and 11). Bars correspond to the 10-bp cutting patterns of DNase I. Sequencing reactions (T; lanes 3 and 12) were run in parallel to precisely map the sites of MNase cuts.
Figure Legend Snippet: MNase accessibility assay of histone–NF-YB–NF-YC combinations. Stoichiometric amounts of the indicated combinations of HFM proteins were reconstituted with fragment 2 (labeled on the top strand [lanes 4 to 9] and bottom strand [lanes 13 to 18]), cut with MNase, and analyzed on sequencing gels. In lane 17, the NF-Y trimer was used to show the NF-Y footprinted area. F refers to free, mock-reconstituted DNA (lanes 4 and 5; uncut and cut with MNase, respectively). Arrows correspond to the major and minor hypersensitive sites. Part of the H3-H4 and H3–H4–NF-YB–NF-YC reconstitutions were cut with DNase I and run in parallel (lanes 1, 2, 10, and 11). Bars correspond to the 10-bp cutting patterns of DNase I. Sequencing reactions (T; lanes 3 and 12) were run in parallel to precisely map the sites of MNase cuts.

Techniques Used: Labeling, Sequencing

11) Product Images from "Persistence of an alternate chromatin structure at silenced loci in the absence of silencers"

Article Title: Persistence of an alternate chromatin structure at silenced loci in the absence of silencers

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

MNase footprinting of chromatin templates. Spheroplasts were made from strains THC27 ( SIR ) and THC28 ( sir3 ), and DNA was digested in situ with MNase (see Materials and Methods ). The indirect end-labeling probe, a LYS2 fragment, hybridizes within the rKWD50N excision cassette adjacent to the Stu I site (these strains lack the LYS2 gene). The positions of HMR E, RS sites, and size markers (in kb) are denoted. (•) identifies a single hypersensitive site outside the excision cassette that appears upon derepression. Digestion of purified chromosomal DNA is also shown (marked Naked). Units of MNase/ml used in each lane: ( Left and Center ) 160, 80, 40, and 20; ( Right ) 10 and 5.
Figure Legend Snippet: MNase footprinting of chromatin templates. Spheroplasts were made from strains THC27 ( SIR ) and THC28 ( sir3 ), and DNA was digested in situ with MNase (see Materials and Methods ). The indirect end-labeling probe, a LYS2 fragment, hybridizes within the rKWD50N excision cassette adjacent to the Stu I site (these strains lack the LYS2 gene). The positions of HMR E, RS sites, and size markers (in kb) are denoted. (•) identifies a single hypersensitive site outside the excision cassette that appears upon derepression. Digestion of purified chromosomal DNA is also shown (marked Naked). Units of MNase/ml used in each lane: ( Left and Center ) 160, 80, 40, and 20; ( Right ) 10 and 5.

Techniques Used: Footprinting, In Situ, End Labeling, Purification

Related Articles

Clone Assay:

Article Title: DNA sequence- and conformation-directed positioning of nucleosomes by chromatin-remodeling complexes
Article Snippet: The 40-bp fragment encompassing the major DNA bending peak (CTGGGGAGGT GGCCCCAAAA ATGACCCCAT AACGAAAAGA) of this DNA was cloned into the pT7 blue3 Vector. .. For mapping the nucleosome positions, 1.5 units MNase (Sigma–Aldrich, St. Louis, MO) were added for 40 sec to remodeling reactions.

Centrifugation:

Article Title: The core spliceosome as target and effector of non-canonical ATM signaling
Article Snippet: .. Following centrifugation (650g, 5 min), nuclei depleted from soluble nucleoplasm were washed with MNase digestion buffer [50 mM Tris-Cl pH 7.5, 4 mM MgCl2 , 50 mM KCl, 300 mM Sucrose, 0.5 mM Na2 VO4 , 5mM NaF, 5mM Na4 P2 O7 , 10mM beta-glycerolphosphate, 1mM PMSF, 1mM, EGTA and 1× EDTA-free Protease inhibitor cocktail] and subsequently incubated with 0.3 U MNase (Sigma)/1×106 nuclei, and 1 mM CaCl2 (37° C, 10 min). .. Addition of (NH4 )2 SO4 to a final concentration of 250 mM was used to facilitate extraction of stably DNA-bound proteins.

Article Title: Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y
Article Snippet: All centrifugation steps before MNase treatment were performed at 3,200 g . .. Mononucleosomes were generated by digestion of chromatin with 0.25 U MNase (Sigma-Aldrich) for 15 min in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 , 0.34 M sucrose, 10% glycerol [vol/vol], 1 mM DTT, and protease inhibitor cocktail [Roche] plus 1 mM CaCl2 ) and stopped by the addition of EGTA (final concentration of 2 mM).

Article Title: Histone octamer trans-transfer: a signature mechanism of ATP-dependent chromatin remodelling unravelled in wheat nuclear extract
Article Snippet: Approximately 0·03 U MNase (Sigma-Aldrich, India) per milligram of DNA was added to the resuspended nuclei and incubated on ice for 2 min with intermittent mixing at an interval of 30 s. Nuclei were centrifuged at 8500 g for 5 min and the pellet was resuspended in nuclei digestion buffer containing 2 m m CaCl2 and the suspension was incubated at 37 °C in a water bath with continuous stirring for a further 2 min. EDTA was added at 0·5 m to stop MNase activity. .. The pellet obtained after centrifugation at 8500 g at 4 °C was resuspended in 15 m m Tris/HCl, pH 7·5, with 0·2 m m EDTA and incubated for 30 min on ice.

Article Title: Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway
Article Snippet: .. After centrifugation (800 × g for 5 min), the nucleus/chromatin fraction in the pellet was re-suspended in 1 ml of hypotonic buffer (10 mM Hepes-NaOH, pH 7.9, 0.25 M potassium acetate, 1 mM dithiothreitol, 0.1% Triton X-100 and protease inhibitors) containing 10 mM CaCl2 and microccocal nuclease (0.2 units/μl; SIGMA, cat. N3755) for 20 min at 37°C. .. Reaction was stopped by adding EGTA (2 mM final concentration) to the sample.

Amplification:

Article Title: Distinct roles for S. cerevisiae H2A copies in recombination and repeat stability, with a role for H2A.1 threonine 126
Article Snippet: .. Mononucleosome positioning detection by Illumina bead array Chromatin was isolated and MNase digested as described above, except mononucleosomes were prepared by digesting the chromatin with 10 units of MNase for 15 min. Purified mononucleosomal DNA was amplified using the GenomePlex Whole Genome Amplification kit (Sigma). .. Amplification and ~150 bp fragment sizes were verified by running in 1.5% agarose.

Whole Genome Amplification:

Article Title: Distinct roles for S. cerevisiae H2A copies in recombination and repeat stability, with a role for H2A.1 threonine 126
Article Snippet: .. Mononucleosome positioning detection by Illumina bead array Chromatin was isolated and MNase digested as described above, except mononucleosomes were prepared by digesting the chromatin with 10 units of MNase for 15 min. Purified mononucleosomal DNA was amplified using the GenomePlex Whole Genome Amplification kit (Sigma). .. Amplification and ~150 bp fragment sizes were verified by running in 1.5% agarose.

Stable Transfection:

Article Title: The core spliceosome as target and effector of non-canonical ATM signaling
Article Snippet: Following centrifugation (650g, 5 min), nuclei depleted from soluble nucleoplasm were washed with MNase digestion buffer [50 mM Tris-Cl pH 7.5, 4 mM MgCl2 , 50 mM KCl, 300 mM Sucrose, 0.5 mM Na2 VO4 , 5mM NaF, 5mM Na4 P2 O7 , 10mM beta-glycerolphosphate, 1mM PMSF, 1mM, EGTA and 1× EDTA-free Protease inhibitor cocktail] and subsequently incubated with 0.3 U MNase (Sigma)/1×106 nuclei, and 1 mM CaCl2 (37° C, 10 min). .. Addition of (NH4 )2 SO4 to a final concentration of 250 mM was used to facilitate extraction of stably DNA-bound proteins.

Blocking Assay:

Article Title: Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway
Article Snippet: Immunoprecipitations were carried out on nuclear extracts prepared from HEK 293T cells synchronized in S-phase with a single block in thymidine. .. After centrifugation (800 × g for 5 min), the nucleus/chromatin fraction in the pellet was re-suspended in 1 ml of hypotonic buffer (10 mM Hepes-NaOH, pH 7.9, 0.25 M potassium acetate, 1 mM dithiothreitol, 0.1% Triton X-100 and protease inhibitors) containing 10 mM CaCl2 and microccocal nuclease (0.2 units/μl; SIGMA, cat. N3755) for 20 min at 37°C.

Electrophoresis:

Article Title: WSTF regulates the function of H2A.X via a novel tyrosine kinase activity
Article Snippet: .. Chromatin pellets were briefly digested with Mnase (Sigma) and the generation of mononucleosomes was monitored by electrophoresis and subject to MS analysis. .. Generation of recombinant WSTF proteins Recombinant baculovirus carrying the full length WSTF (Flag-epitope-tagged at C terminus) was produced using the BaculoGold-BEVS Kit (BD Biosciences).

Article Title: Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway
Article Snippet: Samples were subjected to electrophoresis through 8% polyacrylamide-bis (29:1) gel. .. After centrifugation (800 × g for 5 min), the nucleus/chromatin fraction in the pellet was re-suspended in 1 ml of hypotonic buffer (10 mM Hepes-NaOH, pH 7.9, 0.25 M potassium acetate, 1 mM dithiothreitol, 0.1% Triton X-100 and protease inhibitors) containing 10 mM CaCl2 and microccocal nuclease (0.2 units/μl; SIGMA, cat. N3755) for 20 min at 37°C.

Incubation:

Article Title: The core spliceosome as target and effector of non-canonical ATM signaling
Article Snippet: .. Following centrifugation (650g, 5 min), nuclei depleted from soluble nucleoplasm were washed with MNase digestion buffer [50 mM Tris-Cl pH 7.5, 4 mM MgCl2 , 50 mM KCl, 300 mM Sucrose, 0.5 mM Na2 VO4 , 5mM NaF, 5mM Na4 P2 O7 , 10mM beta-glycerolphosphate, 1mM PMSF, 1mM, EGTA and 1× EDTA-free Protease inhibitor cocktail] and subsequently incubated with 0.3 U MNase (Sigma)/1×106 nuclei, and 1 mM CaCl2 (37° C, 10 min). .. Addition of (NH4 )2 SO4 to a final concentration of 250 mM was used to facilitate extraction of stably DNA-bound proteins.

Article Title: DNA sequence- and conformation-directed positioning of nucleosomes by chromatin-remodeling complexes
Article Snippet: Briefly, nucleosomes and DNA were incubated at ratio of ≈1 remodeler complex per 50 nucleosomes for 90 min at 26°C in the presence of 1 mM ATP, and nucleosome positions were analyzed by native PAGE. .. For mapping the nucleosome positions, 1.5 units MNase (Sigma–Aldrich, St. Louis, MO) were added for 40 sec to remodeling reactions.

Article Title: Mouse Rif1 is a regulatory subunit of protein phosphatase 1 (PP1)
Article Snippet: .. MNase digest: chromatin was incubated in MNase buffer with 1 U MNase (Sigma N3755)/100 μl reaction volume at 28 °C for 1,1.5 and 2 h. The reaction was stopped with 5 μl 200 mM EGTA. .. DNAse I digest: chromatin was incubated in DNAse buffer with 50 U DNAse-I (Roche, 04536282001)/100 μl reaction volume for 2 h at 37 °C.

Article Title: The budding yeast Centromere DNA Element II wraps a stable Cse4 hemisome in either orientation in vivo
Article Snippet: Lightly lyticase-treated cells were digested with MNase for 10 min at 37°C using 4U MNase (Sigma–Aldrich, St. Louis, MO). .. OP labeling (2 hr at room temperature and overnight incubation at 4°C) was followed by three 1M sorbitol 0.1% NP-40 washes, then a sample of OP-treated cells was suspended in fresh MNase buffer and digested with MNase.

Article Title: Histone octamer trans-transfer: a signature mechanism of ATP-dependent chromatin remodelling unravelled in wheat nuclear extract
Article Snippet: .. Approximately 0·03 U MNase (Sigma-Aldrich, India) per milligram of DNA was added to the resuspended nuclei and incubated on ice for 2 min with intermittent mixing at an interval of 30 s. Nuclei were centrifuged at 8500 g for 5 min and the pellet was resuspended in nuclei digestion buffer containing 2 m m CaCl2 and the suspension was incubated at 37 °C in a water bath with continuous stirring for a further 2 min. EDTA was added at 0·5 m to stop MNase activity. .. The pellet obtained after centrifugation at 8500 g at 4 °C was resuspended in 15 m m Tris/HCl, pH 7·5, with 0·2 m m EDTA and incubated for 30 min on ice.

Article Title: Synergy between histone deacetylase inhibitors and DNA-damaging agents is mediated by histone deacetylase 2 in colorectal cancer
Article Snippet: .. A total of 0.06 units of MNase (Sigma-Aldrich, UK) was added to each sample and incubated at 15-20°C for 5 minutes. .. The reaction was stopped by the addition of MNase digestion buffer, MNase stop buffer ((0.5 ml) - 5% SDS; 250 mM EDTA), proteinase K and 20% SDS followed by overnight incubation.

Article Title: Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway
Article Snippet: Where indicated, cells were treated with HU at 2 mM for 9 h. Cells were re-suspended in 1 ml of osmotic buffer (10 mM Hepes-NaOH, pH 7.9, 0.2 M potassium acetate, 0.34 M sucrose, 10% glycerol, 1 mM dithiotreitol, 0.1% Triton X-100 and protease inhibitors) and the sample was incubated for 5 min on ice. .. After centrifugation (800 × g for 5 min), the nucleus/chromatin fraction in the pellet was re-suspended in 1 ml of hypotonic buffer (10 mM Hepes-NaOH, pH 7.9, 0.25 M potassium acetate, 1 mM dithiothreitol, 0.1% Triton X-100 and protease inhibitors) containing 10 mM CaCl2 and microccocal nuclease (0.2 units/μl; SIGMA, cat. N3755) for 20 min at 37°C.

Activity Assay:

Article Title: Histone octamer trans-transfer: a signature mechanism of ATP-dependent chromatin remodelling unravelled in wheat nuclear extract
Article Snippet: .. Approximately 0·03 U MNase (Sigma-Aldrich, India) per milligram of DNA was added to the resuspended nuclei and incubated on ice for 2 min with intermittent mixing at an interval of 30 s. Nuclei were centrifuged at 8500 g for 5 min and the pellet was resuspended in nuclei digestion buffer containing 2 m m CaCl2 and the suspension was incubated at 37 °C in a water bath with continuous stirring for a further 2 min. EDTA was added at 0·5 m to stop MNase activity. .. The pellet obtained after centrifugation at 8500 g at 4 °C was resuspended in 15 m m Tris/HCl, pH 7·5, with 0·2 m m EDTA and incubated for 30 min on ice.

Mass Spectrometry:

Article Title: WSTF regulates the function of H2A.X via a novel tyrosine kinase activity
Article Snippet: .. Chromatin pellets were briefly digested with Mnase (Sigma) and the generation of mononucleosomes was monitored by electrophoresis and subject to MS analysis. .. Generation of recombinant WSTF proteins Recombinant baculovirus carrying the full length WSTF (Flag-epitope-tagged at C terminus) was produced using the BaculoGold-BEVS Kit (BD Biosciences).

Cell Fractionation:

Article Title: Mouse Rif1 is a regulatory subunit of protein phosphatase 1 (PP1)
Article Snippet: ESCs chromatin isolation and Rif1 solubilisation test Cell fractionation and solubilisation of chromatin-bound proteins by salt extraction was performed as described in ref. . .. MNase digest: chromatin was incubated in MNase buffer with 1 U MNase (Sigma N3755)/100 μl reaction volume at 28 °C for 1,1.5 and 2 h. The reaction was stopped with 5 μl 200 mM EGTA.

Modification:

Article Title: The core spliceosome as target and effector of non-canonical ATM signaling
Article Snippet: Following centrifugation (650g, 5 min), nuclei depleted from soluble nucleoplasm were washed with MNase digestion buffer [50 mM Tris-Cl pH 7.5, 4 mM MgCl2 , 50 mM KCl, 300 mM Sucrose, 0.5 mM Na2 VO4 , 5mM NaF, 5mM Na4 P2 O7 , 10mM beta-glycerolphosphate, 1mM PMSF, 1mM, EGTA and 1× EDTA-free Protease inhibitor cocktail] and subsequently incubated with 0.3 U MNase (Sigma)/1×106 nuclei, and 1 mM CaCl2 (37° C, 10 min). .. Protein concentrations were determined using a modified Bradford method (Bio-Rad).

Article Title: The budding yeast Centromere DNA Element II wraps a stable Cse4 hemisome in either orientation in vivo
Article Snippet: Paragraph title: Modified MNase-seq and native ChIP ... Lightly lyticase-treated cells were digested with MNase for 10 min at 37°C using 4U MNase (Sigma–Aldrich, St. Louis, MO).

Western Blot:

Article Title: Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway
Article Snippet: The gel was electro-blotted onto a PVDF membrane and proteins on the blot were detected with a monoclonal horseradish peroxidase-conjugated anti-Flag antibody (Abcam, ab49763) detected using the ECL+ system (GE Healthcare). .. After centrifugation (800 × g for 5 min), the nucleus/chromatin fraction in the pellet was re-suspended in 1 ml of hypotonic buffer (10 mM Hepes-NaOH, pH 7.9, 0.25 M potassium acetate, 1 mM dithiothreitol, 0.1% Triton X-100 and protease inhibitors) containing 10 mM CaCl2 and microccocal nuclease (0.2 units/μl; SIGMA, cat. N3755) for 20 min at 37°C.

Derivative Assay:

Article Title: The budding yeast Centromere DNA Element II wraps a stable Cse4 hemisome in either orientation in vivo
Article Snippet: Samples derived from 500 ml of cells at 2 × 107 /ml were split in half, and one half of each was washed with 1M sorbitol 0.1% NP-40 for OP labeling, while the other other half was suspended in 4 ml fresh MNase buffer (1 M sorbitol; 10 mM Tris-HCl, pH 7.5; 50 mM NaCl; 5 mM MgCl2 ; 2 mM CaCl2 ; 1 mM β-mercaptoethanol, 1 mM phenylmethanesulfonyl fluoride, + 1 protease inhibitor tablet [Roche, Nutley, NJ #04693159001] per 10 ml). .. Lightly lyticase-treated cells were digested with MNase for 10 min at 37°C using 4U MNase (Sigma–Aldrich, St. Louis, MO).

Concentration Assay:

Article Title: The core spliceosome as target and effector of non-canonical ATM signaling
Article Snippet: Following centrifugation (650g, 5 min), nuclei depleted from soluble nucleoplasm were washed with MNase digestion buffer [50 mM Tris-Cl pH 7.5, 4 mM MgCl2 , 50 mM KCl, 300 mM Sucrose, 0.5 mM Na2 VO4 , 5mM NaF, 5mM Na4 P2 O7 , 10mM beta-glycerolphosphate, 1mM PMSF, 1mM, EGTA and 1× EDTA-free Protease inhibitor cocktail] and subsequently incubated with 0.3 U MNase (Sigma)/1×106 nuclei, and 1 mM CaCl2 (37° C, 10 min). .. Addition of (NH4 )2 SO4 to a final concentration of 250 mM was used to facilitate extraction of stably DNA-bound proteins.

Article Title: Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y
Article Snippet: .. Mononucleosomes were generated by digestion of chromatin with 0.25 U MNase (Sigma-Aldrich) for 15 min in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 , 0.34 M sucrose, 10% glycerol [vol/vol], 1 mM DTT, and protease inhibitor cocktail [Roche] plus 1 mM CaCl2 ) and stopped by the addition of EGTA (final concentration of 2 mM). .. Centrifugation was performed at 20,000 g for 20 min. Supernatants of four MNase digests were combined, and salt concentration was adjusted to 150 mM KCl.

Article Title: Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway
Article Snippet: After centrifugation (800 × g for 5 min), the nucleus/chromatin fraction in the pellet was re-suspended in 1 ml of hypotonic buffer (10 mM Hepes-NaOH, pH 7.9, 0.25 M potassium acetate, 1 mM dithiothreitol, 0.1% Triton X-100 and protease inhibitors) containing 10 mM CaCl2 and microccocal nuclease (0.2 units/μl; SIGMA, cat. N3755) for 20 min at 37°C. .. Reaction was stopped by adding EGTA (2 mM final concentration) to the sample.

Protease Inhibitor:

Article Title: The core spliceosome as target and effector of non-canonical ATM signaling
Article Snippet: .. Following centrifugation (650g, 5 min), nuclei depleted from soluble nucleoplasm were washed with MNase digestion buffer [50 mM Tris-Cl pH 7.5, 4 mM MgCl2 , 50 mM KCl, 300 mM Sucrose, 0.5 mM Na2 VO4 , 5mM NaF, 5mM Na4 P2 O7 , 10mM beta-glycerolphosphate, 1mM PMSF, 1mM, EGTA and 1× EDTA-free Protease inhibitor cocktail] and subsequently incubated with 0.3 U MNase (Sigma)/1×106 nuclei, and 1 mM CaCl2 (37° C, 10 min). .. Addition of (NH4 )2 SO4 to a final concentration of 250 mM was used to facilitate extraction of stably DNA-bound proteins.

Article Title: Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y
Article Snippet: .. Mononucleosomes were generated by digestion of chromatin with 0.25 U MNase (Sigma-Aldrich) for 15 min in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 , 0.34 M sucrose, 10% glycerol [vol/vol], 1 mM DTT, and protease inhibitor cocktail [Roche] plus 1 mM CaCl2 ) and stopped by the addition of EGTA (final concentration of 2 mM). .. Centrifugation was performed at 20,000 g for 20 min. Supernatants of four MNase digests were combined, and salt concentration was adjusted to 150 mM KCl.

Article Title: The budding yeast Centromere DNA Element II wraps a stable Cse4 hemisome in either orientation in vivo
Article Snippet: Samples derived from 500 ml of cells at 2 × 107 /ml were split in half, and one half of each was washed with 1M sorbitol 0.1% NP-40 for OP labeling, while the other other half was suspended in 4 ml fresh MNase buffer (1 M sorbitol; 10 mM Tris-HCl, pH 7.5; 50 mM NaCl; 5 mM MgCl2 ; 2 mM CaCl2 ; 1 mM β-mercaptoethanol, 1 mM phenylmethanesulfonyl fluoride, + 1 protease inhibitor tablet [Roche, Nutley, NJ #04693159001] per 10 ml). .. Lightly lyticase-treated cells were digested with MNase for 10 min at 37°C using 4U MNase (Sigma–Aldrich, St. Louis, MO).

Buffer Exchange:

Article Title: Chromatin assembly factor I and Hir proteins contribute to building functional kinetochores in S. cerevisiae
Article Snippet: Pellets were placed facing inward during spins so that centrifugal force mediated buffer exchange in the pellet. .. For indirect end-labeling experiments, nuclei were digested with 0.6 U/mL MNase (Sigma) at 32°C for 0, 5, 10, and 15 min. Digestions were terminated by the addition of one-quarter volume 5% SDS, 20 mM EDTA (pH 8.0), 20 mM EGTA (pH 8.0).

Footprinting:

Article Title: NF-Y Associates with H3-H4 Tetramers and Octamers by Multiple Mechanisms
Article Snippet: Paragraph title: DNase I footprinting, MNase accessibility assay, and Exo III digestion. ... The same amounts of reconstituted tetramers or octamers and free DNA were digested in 2 mM CaCl2 with 0.01 U of MNase (Sigma) at room temperature for 2 min. DNase I and MNase digestions were terminated by adding 2 volumes of DNase I stop buffer (1.5% sodium dodecyl sulfate [SDS], 30 mM EDTA, 450 mM sodium acetate [pH 5.2]).

Article Title: Recruitment of the Nucleolar Remodeling Complex NoRC Establishes Ribosomal DNA Silencing in Chromatin
Article Snippet: Paragraph title: MNase footprinting. ... Purified nucleosomal arrays (300 ng) were digested with 1.5 U of MNase (Sigma) for 40 s. Reactions were conducted in 10 mM Tris-HCl (pH 7.6), 80 mM KCl, 1.5 mM MgCl2 , 10% glycerol, 0.5 mM ATP, and 200 ng of bovine serum albumin per μl and were then stopped by the addition of 0.2 volumes of stop buffer (4% sodium dodecyl sulfate [SDS], 100 mM EDTA, 1 μg of glycogen, 10 μg of proteinase K).

Cell Culture:

Article Title: The budding yeast Centromere DNA Element II wraps a stable Cse4 hemisome in either orientation in vivo
Article Snippet: Modified MNase-seq and native ChIP Wild-type and H4S47C cells were cultured, harvested, lyticased and washed twice in 1M sorbitol 0.1% NP-40 as described for cleavage mapping ( ). .. Lightly lyticase-treated cells were digested with MNase for 10 min at 37°C using 4U MNase (Sigma–Aldrich, St. Louis, MO).

Generated:

Article Title: DNA sequence- and conformation-directed positioning of nucleosomes by chromatin-remodeling complexes
Article Snippet: From this vector, the 253-bp-long pT-K3 fragment with the insert was generated by PCR. .. For mapping the nucleosome positions, 1.5 units MNase (Sigma–Aldrich, St. Louis, MO) were added for 40 sec to remodeling reactions.

Article Title: Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y
Article Snippet: .. Mononucleosomes were generated by digestion of chromatin with 0.25 U MNase (Sigma-Aldrich) for 15 min in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 , 0.34 M sucrose, 10% glycerol [vol/vol], 1 mM DTT, and protease inhibitor cocktail [Roche] plus 1 mM CaCl2 ) and stopped by the addition of EGTA (final concentration of 2 mM). .. Centrifugation was performed at 20,000 g for 20 min. Supernatants of four MNase digests were combined, and salt concentration was adjusted to 150 mM KCl.

Imaging:

Article Title: Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y
Article Snippet: Mononucleosomes were generated by digestion of chromatin with 0.25 U MNase (Sigma-Aldrich) for 15 min in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 , 0.34 M sucrose, 10% glycerol [vol/vol], 1 mM DTT, and protease inhibitor cocktail [Roche] plus 1 mM CaCl2 ) and stopped by the addition of EGTA (final concentration of 2 mM). .. One part was evaluated for DNA size and quality using DNA 1000 reagents (Agilent Technologies) with the 2100 Bioanalyzer (Agilent Technologies), another part was analyzed by immunoblotting using the Odyssey infrared imaging system (LI-COR Biosciences) and evaluated with Odyssey Software Version 2.1 (LI-COR Biosciences), and the last part was analyzed by silver staining.

Sequencing:

Article Title: Recruitment of the Nucleolar Remodeling Complex NoRC Establishes Ribosomal DNA Silencing in Chromatin
Article Snippet: Purified nucleosomal arrays (300 ng) were digested with 1.5 U of MNase (Sigma) for 40 s. Reactions were conducted in 10 mM Tris-HCl (pH 7.6), 80 mM KCl, 1.5 mM MgCl2 , 10% glycerol, 0.5 mM ATP, and 200 ng of bovine serum albumin per μl and were then stopped by the addition of 0.2 volumes of stop buffer (4% sodium dodecyl sulfate [SDS], 100 mM EDTA, 1 μg of glycogen, 10 μg of proteinase K). .. Primer extension fragments were resolved on 8% sequencing gels and quantified with a PhosphorImager and Aida software.

Article Title: DNA sequence encoded repression of rRNA gene transcription in chromatin
Article Snippet: For high resolution mapping of nucleosome positions, purified nucleosomal arrays (300 ng) were digested with 1.5 U of MNase (Sigma) for 40 s. Reactions were conducted in 10 mM Tris–HCl (pH 7.6), 80 mM KCl, 1.5 mM MgCl2 , 10% glycerol, 0.5 mM ATP and 200 ng/μl of bovine serum albumin. .. DNA fragments were resolved on 8% sequencing gels and quantified with a PhosphorImager and Aida software.

Article Title: Distinct roles for S. cerevisiae H2A copies in recombination and repeat stability, with a role for H2A.1 threonine 126
Article Snippet: Mononucleosome positioning detection by Illumina bead array Chromatin was isolated and MNase digested as described above, except mononucleosomes were prepared by digesting the chromatin with 10 units of MNase for 15 min. Purified mononucleosomal DNA was amplified using the GenomePlex Whole Genome Amplification kit (Sigma). .. Purified mononucleosomes were applied to a custom Illumina array that contained YAC CF1 sequence spanning a region 425 bp upstream to 438 bp downstream of the repeat tract in 30 bp non-overlapping probes ( ).

Recombinant:

Article Title: NF-Y Associates with H3-H4 Tetramers and Octamers by Multiple Mechanisms
Article Snippet: For DNase I footprinting, micrococcal nuclease (MNase) accessibility assays, and exonuclease III (Exo III) digestions, we used twice the amount of recombinant histones as employed for EMSA. .. The same amounts of reconstituted tetramers or octamers and free DNA were digested in 2 mM CaCl2 with 0.01 U of MNase (Sigma) at room temperature for 2 min. DNase I and MNase digestions were terminated by adding 2 volumes of DNase I stop buffer (1.5% sodium dodecyl sulfate [SDS], 30 mM EDTA, 450 mM sodium acetate [pH 5.2]).

Sensitive Assay:

Article Title: Synergy between histone deacetylase inhibitors and DNA-damaging agents is mediated by histone deacetylase 2 in colorectal cancer
Article Snippet: Paragraph title: MNase sensitivity assay ... A total of 0.06 units of MNase (Sigma-Aldrich, UK) was added to each sample and incubated at 15-20°C for 5 minutes.

Magnetic Beads:

Article Title: Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y
Article Snippet: Mononucleosomes were generated by digestion of chromatin with 0.25 U MNase (Sigma-Aldrich) for 15 min in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 , 0.34 M sucrose, 10% glycerol [vol/vol], 1 mM DTT, and protease inhibitor cocktail [Roche] plus 1 mM CaCl2 ) and stopped by the addition of EGTA (final concentration of 2 mM). .. Magnetic beads (Invitrogen) were washed four times with buffer C (20 mM Hepes/KOH, pH 7.9, 20% [vol/vol] glycerol, 0.2 mM EDTA, 0.2% [vol/vol] Triton X-100, 300 mM KCl, and protease inhibitor cocktail [Roche]).

Isolation:

Article Title: WSTF regulates the function of H2A.X via a novel tyrosine kinase activity
Article Snippet: Purification of H2A.X-containing mononucleosomes and associated protein factors MEF fractionation and chromatin pellet isolation were performed as described . .. Chromatin pellets were briefly digested with Mnase (Sigma) and the generation of mononucleosomes was monitored by electrophoresis and subject to MS analysis.

Article Title: The core spliceosome as target and effector of non-canonical ATM signaling
Article Snippet: Crude chromatin was isolated after Triton-X 100 extraction and MNase digestion. .. Following centrifugation (650g, 5 min), nuclei depleted from soluble nucleoplasm were washed with MNase digestion buffer [50 mM Tris-Cl pH 7.5, 4 mM MgCl2 , 50 mM KCl, 300 mM Sucrose, 0.5 mM Na2 VO4 , 5mM NaF, 5mM Na4 P2 O7 , 10mM beta-glycerolphosphate, 1mM PMSF, 1mM, EGTA and 1× EDTA-free Protease inhibitor cocktail] and subsequently incubated with 0.3 U MNase (Sigma)/1×106 nuclei, and 1 mM CaCl2 (37° C, 10 min).

Article Title: DNA sequence- and conformation-directed positioning of nucleosomes by chromatin-remodeling complexes
Article Snippet: For mapping the nucleosome positions, 1.5 units MNase (Sigma–Aldrich, St. Louis, MO) were added for 40 sec to remodeling reactions. .. The protected nucleosomal core DNA was isolated and analyzed by a single round of PCR (denaturation, 5 min at 95°C; annealing, 2 min at 56°C; extension, 1 min at 72°C) by using at least three different 32 .

Article Title: Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y
Article Snippet: In brief, nuclei were isolated by hypotonic lysis and extracted using 0.4 M sulfuric acid. .. Mononucleosomes were generated by digestion of chromatin with 0.25 U MNase (Sigma-Aldrich) for 15 min in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 , 0.34 M sucrose, 10% glycerol [vol/vol], 1 mM DTT, and protease inhibitor cocktail [Roche] plus 1 mM CaCl2 ) and stopped by the addition of EGTA (final concentration of 2 mM).

Article Title: Mouse Rif1 is a regulatory subunit of protein phosphatase 1 (PP1)
Article Snippet: Paragraph title: ESCs chromatin isolation and Rif1 solubilisation test ... MNase digest: chromatin was incubated in MNase buffer with 1 U MNase (Sigma N3755)/100 μl reaction volume at 28 °C for 1,1.5 and 2 h. The reaction was stopped with 5 μl 200 mM EGTA.

Article Title: The budding yeast Centromere DNA Element II wraps a stable Cse4 hemisome in either orientation in vivo
Article Snippet: Lightly lyticase-treated cells were digested with MNase for 10 min at 37°C using 4U MNase (Sigma–Aldrich, St. Louis, MO). .. For soluble and insoluble chromatin isolation and ChIP, the remaining samples were snap-frozen for storage, thawed on ice, and soluble chromatin was extracted and ChIP was performed as described ( ).

Article Title: Distinct roles for S. cerevisiae H2A copies in recombination and repeat stability, with a role for H2A.1 threonine 126
Article Snippet: .. Mononucleosome positioning detection by Illumina bead array Chromatin was isolated and MNase digested as described above, except mononucleosomes were prepared by digesting the chromatin with 10 units of MNase for 15 min. Purified mononucleosomal DNA was amplified using the GenomePlex Whole Genome Amplification kit (Sigma). .. Amplification and ~150 bp fragment sizes were verified by running in 1.5% agarose.

Size-exclusion Chromatography:

Article Title: DNA sequence- and conformation-directed positioning of nucleosomes by chromatin-remodeling complexes
Article Snippet: .. For mapping the nucleosome positions, 1.5 units MNase (Sigma–Aldrich, St. Louis, MO) were added for 40 sec to remodeling reactions. .. The protected nucleosomal core DNA was isolated and analyzed by a single round of PCR (denaturation, 5 min at 95°C; annealing, 2 min at 56°C; extension, 1 min at 72°C) by using at least three different 32 .

Labeling:

Article Title: Recruitment of the Nucleolar Remodeling Complex NoRC Establishes Ribosomal DNA Silencing in Chromatin
Article Snippet: Purified nucleosomal arrays (300 ng) were digested with 1.5 U of MNase (Sigma) for 40 s. Reactions were conducted in 10 mM Tris-HCl (pH 7.6), 80 mM KCl, 1.5 mM MgCl2 , 10% glycerol, 0.5 mM ATP, and 200 ng of bovine serum albumin per μl and were then stopped by the addition of 0.2 volumes of stop buffer (4% sodium dodecyl sulfate [SDS], 100 mM EDTA, 1 μg of glycogen, 10 μg of proteinase K). .. DNA was purified and analyzed by a single round of PCR (denaturation, 5 min at 95°C; annealing, 2 min at 56°C; extension, 1 min at 72°C) using radioactively labeled oligonucleotides that hybridized to the rDNA promoter.

Article Title: The budding yeast Centromere DNA Element II wraps a stable Cse4 hemisome in either orientation in vivo
Article Snippet: Samples derived from 500 ml of cells at 2 × 107 /ml were split in half, and one half of each was washed with 1M sorbitol 0.1% NP-40 for OP labeling, while the other other half was suspended in 4 ml fresh MNase buffer (1 M sorbitol; 10 mM Tris-HCl, pH 7.5; 50 mM NaCl; 5 mM MgCl2 ; 2 mM CaCl2 ; 1 mM β-mercaptoethanol, 1 mM phenylmethanesulfonyl fluoride, + 1 protease inhibitor tablet [Roche, Nutley, NJ #04693159001] per 10 ml). .. Lightly lyticase-treated cells were digested with MNase for 10 min at 37°C using 4U MNase (Sigma–Aldrich, St. Louis, MO).

Article Title: Distinct roles for S. cerevisiae H2A copies in recombination and repeat stability, with a role for H2A.1 threonine 126
Article Snippet: Mononucleosome positioning detection by Illumina bead array Chromatin was isolated and MNase digested as described above, except mononucleosomes were prepared by digesting the chromatin with 10 units of MNase for 15 min. Purified mononucleosomal DNA was amplified using the GenomePlex Whole Genome Amplification kit (Sigma). .. Amplified mononuclesomes were purified with a GenElute PCR clean-up kit (Sigma) and 3’ biotin end-labled (Pierce); DNA was chloroform extracted, and labeling was verified by dot blot according to the manufacturer’s instructions.

Purification:

Article Title: WSTF regulates the function of H2A.X via a novel tyrosine kinase activity
Article Snippet: Paragraph title: Purification of H2A.X-containing mononucleosomes and associated protein factors ... Chromatin pellets were briefly digested with Mnase (Sigma) and the generation of mononucleosomes was monitored by electrophoresis and subject to MS analysis.

Article Title: Recruitment of the Nucleolar Remodeling Complex NoRC Establishes Ribosomal DNA Silencing in Chromatin
Article Snippet: .. Purified nucleosomal arrays (300 ng) were digested with 1.5 U of MNase (Sigma) for 40 s. Reactions were conducted in 10 mM Tris-HCl (pH 7.6), 80 mM KCl, 1.5 mM MgCl2 , 10% glycerol, 0.5 mM ATP, and 200 ng of bovine serum albumin per μl and were then stopped by the addition of 0.2 volumes of stop buffer (4% sodium dodecyl sulfate [SDS], 100 mM EDTA, 1 μg of glycogen, 10 μg of proteinase K). .. DNA was purified and analyzed by a single round of PCR (denaturation, 5 min at 95°C; annealing, 2 min at 56°C; extension, 1 min at 72°C) using radioactively labeled oligonucleotides that hybridized to the rDNA promoter.

Article Title: DNA sequence encoded repression of rRNA gene transcription in chromatin
Article Snippet: .. For high resolution mapping of nucleosome positions, purified nucleosomal arrays (300 ng) were digested with 1.5 U of MNase (Sigma) for 40 s. Reactions were conducted in 10 mM Tris–HCl (pH 7.6), 80 mM KCl, 1.5 mM MgCl2 , 10% glycerol, 0.5 mM ATP and 200 ng/μl of bovine serum albumin. ..

Article Title: Chromatin assembly factor I and Hir proteins contribute to building functional kinetochores in S. cerevisiae
Article Snippet: For indirect end-labeling experiments, nuclei were digested with 0.6 U/mL MNase (Sigma) at 32°C for 0, 5, 10, and 15 min. Digestions were terminated by the addition of one-quarter volume 5% SDS, 20 mM EDTA (pH 8.0), 20 mM EGTA (pH 8.0). .. To analyze nucleosome positioning proximal to an 8.5-kb region adjacent to CDEI, genomic DNA from MNase-treated samples was purified and digested with Bam HI.

Article Title: Distinct roles for S. cerevisiae H2A copies in recombination and repeat stability, with a role for H2A.1 threonine 126
Article Snippet: .. Mononucleosome positioning detection by Illumina bead array Chromatin was isolated and MNase digested as described above, except mononucleosomes were prepared by digesting the chromatin with 10 units of MNase for 15 min. Purified mononucleosomal DNA was amplified using the GenomePlex Whole Genome Amplification kit (Sigma). .. Amplification and ~150 bp fragment sizes were verified by running in 1.5% agarose.

Dot Blot:

Article Title: Distinct roles for S. cerevisiae H2A copies in recombination and repeat stability, with a role for H2A.1 threonine 126
Article Snippet: Mononucleosome positioning detection by Illumina bead array Chromatin was isolated and MNase digested as described above, except mononucleosomes were prepared by digesting the chromatin with 10 units of MNase for 15 min. Purified mononucleosomal DNA was amplified using the GenomePlex Whole Genome Amplification kit (Sigma). .. Amplified mononuclesomes were purified with a GenElute PCR clean-up kit (Sigma) and 3’ biotin end-labled (Pierce); DNA was chloroform extracted, and labeling was verified by dot blot according to the manufacturer’s instructions.

Polymerase Chain Reaction:

Article Title: DNA sequence- and conformation-directed positioning of nucleosomes by chromatin-remodeling complexes
Article Snippet: From this vector, the 253-bp-long pT-K3 fragment with the insert was generated by PCR. .. For mapping the nucleosome positions, 1.5 units MNase (Sigma–Aldrich, St. Louis, MO) were added for 40 sec to remodeling reactions.

Article Title: Recruitment of the Nucleolar Remodeling Complex NoRC Establishes Ribosomal DNA Silencing in Chromatin
Article Snippet: Purified nucleosomal arrays (300 ng) were digested with 1.5 U of MNase (Sigma) for 40 s. Reactions were conducted in 10 mM Tris-HCl (pH 7.6), 80 mM KCl, 1.5 mM MgCl2 , 10% glycerol, 0.5 mM ATP, and 200 ng of bovine serum albumin per μl and were then stopped by the addition of 0.2 volumes of stop buffer (4% sodium dodecyl sulfate [SDS], 100 mM EDTA, 1 μg of glycogen, 10 μg of proteinase K). .. DNA was purified and analyzed by a single round of PCR (denaturation, 5 min at 95°C; annealing, 2 min at 56°C; extension, 1 min at 72°C) using radioactively labeled oligonucleotides that hybridized to the rDNA promoter.

Article Title: Chromatin assembly factor I and Hir proteins contribute to building functional kinetochores in S. cerevisiae
Article Snippet: For indirect end-labeling experiments, nuclei were digested with 0.6 U/mL MNase (Sigma) at 32°C for 0, 5, 10, and 15 min. Digestions were terminated by the addition of one-quarter volume 5% SDS, 20 mM EDTA (pH 8.0), 20 mM EGTA (pH 8.0). .. Blots were hybridized using a PCR-generated probe corresponding to SGD coordinates 113757–114293 on Chromosome III.

Article Title: Distinct roles for S. cerevisiae H2A copies in recombination and repeat stability, with a role for H2A.1 threonine 126
Article Snippet: Mononucleosome positioning detection by Illumina bead array Chromatin was isolated and MNase digested as described above, except mononucleosomes were prepared by digesting the chromatin with 10 units of MNase for 15 min. Purified mononucleosomal DNA was amplified using the GenomePlex Whole Genome Amplification kit (Sigma). .. Amplified mononuclesomes were purified with a GenElute PCR clean-up kit (Sigma) and 3’ biotin end-labled (Pierce); DNA was chloroform extracted, and labeling was verified by dot blot according to the manufacturer’s instructions.

Silver Staining:

Article Title: Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y
Article Snippet: Mononucleosomes were generated by digestion of chromatin with 0.25 U MNase (Sigma-Aldrich) for 15 min in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 , 0.34 M sucrose, 10% glycerol [vol/vol], 1 mM DTT, and protease inhibitor cocktail [Roche] plus 1 mM CaCl2 ) and stopped by the addition of EGTA (final concentration of 2 mM). .. One part was evaluated for DNA size and quality using DNA 1000 reagents (Agilent Technologies) with the 2100 Bioanalyzer (Agilent Technologies), another part was analyzed by immunoblotting using the Odyssey infrared imaging system (LI-COR Biosciences) and evaluated with Odyssey Software Version 2.1 (LI-COR Biosciences), and the last part was analyzed by silver staining.

Chromatin Immunoprecipitation:

Article Title: The budding yeast Centromere DNA Element II wraps a stable Cse4 hemisome in either orientation in vivo
Article Snippet: Paragraph title: Modified MNase-seq and native ChIP ... Lightly lyticase-treated cells were digested with MNase for 10 min at 37°C using 4U MNase (Sigma–Aldrich, St. Louis, MO).

SDS Page:

Article Title: Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway
Article Snippet: The beads were then washed three times with 600 μl of washing buffer (25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 300 mM NaCl, 1 mM dithiotreitol, 0.1% Nonidet-P40) and re-suspended in 30 μl of SDS-PAGE loading buffer (50 mM Tris-HCl, pH 6.8, 10% glycerol, 200 mM β-mercaptoethanol, 0.5% SDS, 0.01% blue bromophenol). .. After centrifugation (800 × g for 5 min), the nucleus/chromatin fraction in the pellet was re-suspended in 1 ml of hypotonic buffer (10 mM Hepes-NaOH, pH 7.9, 0.25 M potassium acetate, 1 mM dithiothreitol, 0.1% Triton X-100 and protease inhibitors) containing 10 mM CaCl2 and microccocal nuclease (0.2 units/μl; SIGMA, cat. N3755) for 20 min at 37°C.

Plasmid Preparation:

Article Title: DNA sequence- and conformation-directed positioning of nucleosomes by chromatin-remodeling complexes
Article Snippet: From this vector, the 253-bp-long pT-K3 fragment with the insert was generated by PCR. .. For mapping the nucleosome positions, 1.5 units MNase (Sigma–Aldrich, St. Louis, MO) were added for 40 sec to remodeling reactions.

Software:

Article Title: Recruitment of the Nucleolar Remodeling Complex NoRC Establishes Ribosomal DNA Silencing in Chromatin
Article Snippet: Purified nucleosomal arrays (300 ng) were digested with 1.5 U of MNase (Sigma) for 40 s. Reactions were conducted in 10 mM Tris-HCl (pH 7.6), 80 mM KCl, 1.5 mM MgCl2 , 10% glycerol, 0.5 mM ATP, and 200 ng of bovine serum albumin per μl and were then stopped by the addition of 0.2 volumes of stop buffer (4% sodium dodecyl sulfate [SDS], 100 mM EDTA, 1 μg of glycogen, 10 μg of proteinase K). .. Primer extension fragments were resolved on 8% sequencing gels and quantified with a PhosphorImager and Aida software.

Article Title: Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y
Article Snippet: Mononucleosomes were generated by digestion of chromatin with 0.25 U MNase (Sigma-Aldrich) for 15 min in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 , 0.34 M sucrose, 10% glycerol [vol/vol], 1 mM DTT, and protease inhibitor cocktail [Roche] plus 1 mM CaCl2 ) and stopped by the addition of EGTA (final concentration of 2 mM). .. One part was evaluated for DNA size and quality using DNA 1000 reagents (Agilent Technologies) with the 2100 Bioanalyzer (Agilent Technologies), another part was analyzed by immunoblotting using the Odyssey infrared imaging system (LI-COR Biosciences) and evaluated with Odyssey Software Version 2.1 (LI-COR Biosciences), and the last part was analyzed by silver staining.

Article Title: DNA sequence encoded repression of rRNA gene transcription in chromatin
Article Snippet: For high resolution mapping of nucleosome positions, purified nucleosomal arrays (300 ng) were digested with 1.5 U of MNase (Sigma) for 40 s. Reactions were conducted in 10 mM Tris–HCl (pH 7.6), 80 mM KCl, 1.5 mM MgCl2 , 10% glycerol, 0.5 mM ATP and 200 ng/μl of bovine serum albumin. .. DNA fragments were resolved on 8% sequencing gels and quantified with a PhosphorImager and Aida software.

Ethanol Precipitation:

Article Title: Synergy between histone deacetylase inhibitors and DNA-damaging agents is mediated by histone deacetylase 2 in colorectal cancer
Article Snippet: A total of 0.06 units of MNase (Sigma-Aldrich, UK) was added to each sample and incubated at 15-20°C for 5 minutes. .. DNA was extracted by standard phenol/chloroform extraction and ethanol precipitation.

Immunoprecipitation:

Article Title: Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway
Article Snippet: Paragraph title: Immunoprecipitation experiments ... After centrifugation (800 × g for 5 min), the nucleus/chromatin fraction in the pellet was re-suspended in 1 ml of hypotonic buffer (10 mM Hepes-NaOH, pH 7.9, 0.25 M potassium acetate, 1 mM dithiothreitol, 0.1% Triton X-100 and protease inhibitors) containing 10 mM CaCl2 and microccocal nuclease (0.2 units/μl; SIGMA, cat. N3755) for 20 min at 37°C.

Fractionation:

Article Title: WSTF regulates the function of H2A.X via a novel tyrosine kinase activity
Article Snippet: Purification of H2A.X-containing mononucleosomes and associated protein factors MEF fractionation and chromatin pellet isolation were performed as described . .. Chromatin pellets were briefly digested with Mnase (Sigma) and the generation of mononucleosomes was monitored by electrophoresis and subject to MS analysis.

Article Title: The core spliceosome as target and effector of non-canonical ATM signaling
Article Snippet: Paragraph title: Preparation of whole cell lysates, Chromatin fractionation, and Immunoprecipitations ... Following centrifugation (650g, 5 min), nuclei depleted from soluble nucleoplasm were washed with MNase digestion buffer [50 mM Tris-Cl pH 7.5, 4 mM MgCl2 , 50 mM KCl, 300 mM Sucrose, 0.5 mM Na2 VO4 , 5mM NaF, 5mM Na4 P2 O7 , 10mM beta-glycerolphosphate, 1mM PMSF, 1mM, EGTA and 1× EDTA-free Protease inhibitor cocktail] and subsequently incubated with 0.3 U MNase (Sigma)/1×106 nuclei, and 1 mM CaCl2 (37° C, 10 min).

End Labeling:

Article Title: Chromatin assembly factor I and Hir proteins contribute to building functional kinetochores in S. cerevisiae
Article Snippet: .. For indirect end-labeling experiments, nuclei were digested with 0.6 U/mL MNase (Sigma) at 32°C for 0, 5, 10, and 15 min. Digestions were terminated by the addition of one-quarter volume 5% SDS, 20 mM EDTA (pH 8.0), 20 mM EGTA (pH 8.0). ..

Lysis:

Article Title: The core spliceosome as target and effector of non-canonical ATM signaling
Article Snippet: Preparation of whole cell lysates, Chromatin fractionation, and Immunoprecipitations Whole cell lysates were prepared by lysis of equal cell numbers in 60 mM Tris-Cl pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol and 0.01% bromophenol blue. .. Following centrifugation (650g, 5 min), nuclei depleted from soluble nucleoplasm were washed with MNase digestion buffer [50 mM Tris-Cl pH 7.5, 4 mM MgCl2 , 50 mM KCl, 300 mM Sucrose, 0.5 mM Na2 VO4 , 5mM NaF, 5mM Na4 P2 O7 , 10mM beta-glycerolphosphate, 1mM PMSF, 1mM, EGTA and 1× EDTA-free Protease inhibitor cocktail] and subsequently incubated with 0.3 U MNase (Sigma)/1×106 nuclei, and 1 mM CaCl2 (37° C, 10 min).

Article Title: Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y
Article Snippet: Mononucleosomes for IP experiments were generated as described previously , with the following changes: For cell lysis, 0.1% NP-40 was used instead of Triton X-100. .. Mononucleosomes were generated by digestion of chromatin with 0.25 U MNase (Sigma-Aldrich) for 15 min in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 , 0.34 M sucrose, 10% glycerol [vol/vol], 1 mM DTT, and protease inhibitor cocktail [Roche] plus 1 mM CaCl2 ) and stopped by the addition of EGTA (final concentration of 2 mM).

Article Title: Synergy between histone deacetylase inhibitors and DNA-damaging agents is mediated by histone deacetylase 2 in colorectal cancer
Article Snippet: MNase sensitivity assay Cells were lysed in NP-40 lysis buffer (ce-cold NP-40 lysis buffer (10 mM Tris [pH 7.4], 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40, 0.15 mM spermine, 0.5 mM spermidine) and incubated on ice for 5 minutes.). .. A total of 0.06 units of MNase (Sigma-Aldrich, UK) was added to each sample and incubated at 15-20°C for 5 minutes.

Clear Native PAGE:

Article Title: DNA sequence- and conformation-directed positioning of nucleosomes by chromatin-remodeling complexes
Article Snippet: Briefly, nucleosomes and DNA were incubated at ratio of ≈1 remodeler complex per 50 nucleosomes for 90 min at 26°C in the presence of 1 mM ATP, and nucleosome positions were analyzed by native PAGE. .. For mapping the nucleosome positions, 1.5 units MNase (Sigma–Aldrich, St. Louis, MO) were added for 40 sec to remodeling reactions.

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    Millipore micrococcal nuclease mnase
    Micrococcal Nuclease Mnase, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Millipore mnase digested chip dna
    Scheme of the EBV genome and experimental design for the analyses of pre-RC and SNS zones as well as mapping of MR profiles. (A) Scheme of the circular EBV genome. In addition to the latent origins oriP (blue box) and the 14-kbp “Raji origin” (blue line), the lytic origin (oriLyt) is shown. The latent EBV nuclear antigens 1, 2, 3A–C, EBNA-LP genes (turquoise), and LMP1 and -2 (purple) are depicted, including their transcripts and promoters. The EBER1 and -2 and the miRNAs regions (BART and BARF) are indicated (green lines). The Raji genome harbors two deletions (red Δ, nt 86,000–89,000 and 163,978–166,635). These regions do not produce array signals in comparison to the reference strain type I used for the design of the EBV microarray. (B) Chart of the experimental set up to map pre-RC zones (left), <t>MNase</t> profiles (central), and SNS <t>DNA</t> (right). (C) Cell cycle phases of logarithmically growing Raji cells were separated by centrifugal elutriation. The DNA content of the different fractions was determined by FACS analysis (top, I–VI). The FACS profiles of one out of three experiments are shown. The quality of coprecipitated DNA was determined by quantitative PCR. The histograms show the mean values of three independent Orc2 (bottom left) and Mcm3 (bottom right) immunoprecipitations. The enrichments of Orc2 (red bars) and Mcm3 enrichments (blue bars) at the DS region are shown. The black bars indicate the enrichments of Orc2 and Mcm3 to a reference site. Error bars indicate mean ± SEM.
    Mnase Digested Chip Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mnase digested chip dna/product/Millipore
    Average 79 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mnase digested chip dna - by Bioz Stars, 2020-01
    79/100 stars
      Buy from Supplier

    78
    Millipore mnase1
    Scheme of the EBV genome and experimental design for the analyses of pre-RC and SNS zones as well as mapping of MR profiles. (A) Scheme of the circular EBV genome. In addition to the latent origins oriP (blue box) and the 14-kbp “Raji origin” (blue line), the lytic origin (oriLyt) is shown. The latent EBV nuclear antigens 1, 2, 3A–C, EBNA-LP genes (turquoise), and LMP1 and -2 (purple) are depicted, including their transcripts and promoters. The EBER1 and -2 and the miRNAs regions (BART and BARF) are indicated (green lines). The Raji genome harbors two deletions (red Δ, nt 86,000–89,000 and 163,978–166,635). These regions do not produce array signals in comparison to the reference strain type I used for the design of the EBV microarray. (B) Chart of the experimental set up to map pre-RC zones (left), <t>MNase</t> profiles (central), and SNS <t>DNA</t> (right). (C) Cell cycle phases of logarithmically growing Raji cells were separated by centrifugal elutriation. The DNA content of the different fractions was determined by FACS analysis (top, I–VI). The FACS profiles of one out of three experiments are shown. The quality of coprecipitated DNA was determined by quantitative PCR. The histograms show the mean values of three independent Orc2 (bottom left) and Mcm3 (bottom right) immunoprecipitations. The enrichments of Orc2 (red bars) and Mcm3 enrichments (blue bars) at the DS region are shown. The black bars indicate the enrichments of Orc2 and Mcm3 to a reference site. Error bars indicate mean ± SEM.
    Mnase1, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mnase1/product/Millipore
    Average 78 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    mnase1 - by Bioz Stars, 2020-01
    78/100 stars
      Buy from Supplier

    Image Search Results


    Scheme of the EBV genome and experimental design for the analyses of pre-RC and SNS zones as well as mapping of MR profiles. (A) Scheme of the circular EBV genome. In addition to the latent origins oriP (blue box) and the 14-kbp “Raji origin” (blue line), the lytic origin (oriLyt) is shown. The latent EBV nuclear antigens 1, 2, 3A–C, EBNA-LP genes (turquoise), and LMP1 and -2 (purple) are depicted, including their transcripts and promoters. The EBER1 and -2 and the miRNAs regions (BART and BARF) are indicated (green lines). The Raji genome harbors two deletions (red Δ, nt 86,000–89,000 and 163,978–166,635). These regions do not produce array signals in comparison to the reference strain type I used for the design of the EBV microarray. (B) Chart of the experimental set up to map pre-RC zones (left), MNase profiles (central), and SNS DNA (right). (C) Cell cycle phases of logarithmically growing Raji cells were separated by centrifugal elutriation. The DNA content of the different fractions was determined by FACS analysis (top, I–VI). The FACS profiles of one out of three experiments are shown. The quality of coprecipitated DNA was determined by quantitative PCR. The histograms show the mean values of three independent Orc2 (bottom left) and Mcm3 (bottom right) immunoprecipitations. The enrichments of Orc2 (red bars) and Mcm3 enrichments (blue bars) at the DS region are shown. The black bars indicate the enrichments of Orc2 and Mcm3 to a reference site. Error bars indicate mean ± SEM.

    Journal: The Journal of Cell Biology

    Article Title: Open chromatin structures regulate the efficiencies of pre-RC formation and replication initiation in Epstein-Barr virus

    doi: 10.1083/jcb.201109105

    Figure Lengend Snippet: Scheme of the EBV genome and experimental design for the analyses of pre-RC and SNS zones as well as mapping of MR profiles. (A) Scheme of the circular EBV genome. In addition to the latent origins oriP (blue box) and the 14-kbp “Raji origin” (blue line), the lytic origin (oriLyt) is shown. The latent EBV nuclear antigens 1, 2, 3A–C, EBNA-LP genes (turquoise), and LMP1 and -2 (purple) are depicted, including their transcripts and promoters. The EBER1 and -2 and the miRNAs regions (BART and BARF) are indicated (green lines). The Raji genome harbors two deletions (red Δ, nt 86,000–89,000 and 163,978–166,635). These regions do not produce array signals in comparison to the reference strain type I used for the design of the EBV microarray. (B) Chart of the experimental set up to map pre-RC zones (left), MNase profiles (central), and SNS DNA (right). (C) Cell cycle phases of logarithmically growing Raji cells were separated by centrifugal elutriation. The DNA content of the different fractions was determined by FACS analysis (top, I–VI). The FACS profiles of one out of three experiments are shown. The quality of coprecipitated DNA was determined by quantitative PCR. The histograms show the mean values of three independent Orc2 (bottom left) and Mcm3 (bottom right) immunoprecipitations. The enrichments of Orc2 (red bars) and Mcm3 enrichments (blue bars) at the DS region are shown. The black bars indicate the enrichments of Orc2 and Mcm3 to a reference site. Error bars indicate mean ± SEM.

    Article Snippet: Southern blotting 500 ng of sonicated and MNase-digested ChIP DNA or nascent strand DNA were separated on a 1.0% TAE gel and transferred to membrane (Immobilon Ny+; EMD Millipore).

    Techniques: Microarray, FACS, Real-time Polymerase Chain Reaction