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Frequencies of occurrence of DNA dinucleotide steps in the +1 <t>nucleosomes</t> of yeast and the sketch of <t>MNase-seq</t> experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.
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Images

1) Product Images from "MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast"

Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky502

Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.
Figure Legend Snippet: Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.

Techniques Used:

MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.
Figure Legend Snippet: MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.

Techniques Used: Incubation, Atomic Absorption Spectroscopy

Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).
Figure Legend Snippet: Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).

Techniques Used: Sequencing

2) Product Images from "Crystal structure and stable property of the cancer-associated heterotypic nucleosome containing CENP-A and H3.3"

Article Title: Crystal structure and stable property of the cancer-associated heterotypic nucleosome containing CENP-A and H3.3

Journal: Scientific Reports

doi: 10.1038/srep07115

The DNA end close to CENP-A is asymmetrically flexible in the CENP-A/H3.3 nucleosome. (a) MNase assay. The H3.3, CENP-A, and CENP-A/H3.3 nucleosomes were treated with MNase (0, 0.3, 0.5 and 0.7 units), and the resulting DNA fragments were analyzed by native PAGE. The gel image shown is a representative of four independent experiments, in which similar results were obtained. (b) ExoIII assay. The H3.3, CENP-A, or CENP-A/H3.3 nucleosomes were incubated with or without 2.0 units of ExoIII for 2.5, 5 and 7.5 minutes at 37°C, and the resultant DNA fragments were extracted and analyzed by 14% denaturing PAGE with 7 M urea. The gel image shown is a representative of three independent experiments, in which similar results were obtained.
Figure Legend Snippet: The DNA end close to CENP-A is asymmetrically flexible in the CENP-A/H3.3 nucleosome. (a) MNase assay. The H3.3, CENP-A, and CENP-A/H3.3 nucleosomes were treated with MNase (0, 0.3, 0.5 and 0.7 units), and the resulting DNA fragments were analyzed by native PAGE. The gel image shown is a representative of four independent experiments, in which similar results were obtained. (b) ExoIII assay. The H3.3, CENP-A, or CENP-A/H3.3 nucleosomes were incubated with or without 2.0 units of ExoIII for 2.5, 5 and 7.5 minutes at 37°C, and the resultant DNA fragments were extracted and analyzed by 14% denaturing PAGE with 7 M urea. The gel image shown is a representative of three independent experiments, in which similar results were obtained.

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

The DNA end close to CENP-A is asymmetrically flexible in the CENP-A/H3.3 nucleosome. (a) MNase assay. The H3.3, CENP-A, and CENP-A/H3.3 nucleosomes were treated with MNase (0, 0.3, 0.5 and 0.7 units), and the resulting DNA fragments were analyzed by native PAGE. The gel image shown is a representative of four independent experiments, in which similar results were obtained. (b) ExoIII assay. The H3.3, CENP-A, or CENP-A/H3.3 nucleosomes were incubated with or without 2.0 units of ExoIII for 2.5, 5 and 7.5 minutes at 37°C, and the resultant DNA fragments were extracted and analyzed by 14% denaturing PAGE with 7 M urea. The gel image shown is a representative of three independent experiments, in which similar results were obtained.
Figure Legend Snippet: The DNA end close to CENP-A is asymmetrically flexible in the CENP-A/H3.3 nucleosome. (a) MNase assay. The H3.3, CENP-A, and CENP-A/H3.3 nucleosomes were treated with MNase (0, 0.3, 0.5 and 0.7 units), and the resulting DNA fragments were analyzed by native PAGE. The gel image shown is a representative of four independent experiments, in which similar results were obtained. (b) ExoIII assay. The H3.3, CENP-A, or CENP-A/H3.3 nucleosomes were incubated with or without 2.0 units of ExoIII for 2.5, 5 and 7.5 minutes at 37°C, and the resultant DNA fragments were extracted and analyzed by 14% denaturing PAGE with 7 M urea. The gel image shown is a representative of three independent experiments, in which similar results were obtained.

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

3) Product Images from "MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast"

Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky502

Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.
Figure Legend Snippet: Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.

Techniques Used:

MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.
Figure Legend Snippet: MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.

Techniques Used: Incubation, Atomic Absorption Spectroscopy

Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).
Figure Legend Snippet: Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).

Techniques Used: Sequencing

Correlation between MNase digestions and contents of site-exposure sequence by comparing the two ends of a nucleosome. Correlations between MNase digestions and AA/TT contents in the 1- and 3-min assays are shown on the left and right panels of ( A ), respectively. Similarly, ( B ) shows the correlations of MNase digestion versus AAAA/TTTT content from the 1- and 3-min assays. The shaded rectangle regions in red indicate that the entry site of a nucleosome with more AA/TTs or AAAA/TTTTs gets more digested; the regions in blue indicate that the exit site with more AA/TTs or AAAA/TTTTs gets more digested. The 20 +1 nucleosomes are divided into two groups based on the numbers of s ite e xposure s equence e lements (SESEs, defined as discrete AAAA or TTTT segments) in their sequences. Specifically, nucleosomes with SESEs (coloured in black) are those with three or more SESEs on either strand of the nucleosomes, including nuc01, nuc02, nuc03, nuc05, nuc07, nuc10 and nuc20. Oppositely, nucleosomes with no or fewer SESEs (coloured in orange) consisting of the rest of the +1 nucleosomes, are those with two or fewer SESEs on each strand. Correlation coefficients for each class of nucleosomes under each incubation time are also indicated.
Figure Legend Snippet: Correlation between MNase digestions and contents of site-exposure sequence by comparing the two ends of a nucleosome. Correlations between MNase digestions and AA/TT contents in the 1- and 3-min assays are shown on the left and right panels of ( A ), respectively. Similarly, ( B ) shows the correlations of MNase digestion versus AAAA/TTTT content from the 1- and 3-min assays. The shaded rectangle regions in red indicate that the entry site of a nucleosome with more AA/TTs or AAAA/TTTTs gets more digested; the regions in blue indicate that the exit site with more AA/TTs or AAAA/TTTTs gets more digested. The 20 +1 nucleosomes are divided into two groups based on the numbers of s ite e xposure s equence e lements (SESEs, defined as discrete AAAA or TTTT segments) in their sequences. Specifically, nucleosomes with SESEs (coloured in black) are those with three or more SESEs on either strand of the nucleosomes, including nuc01, nuc02, nuc03, nuc05, nuc07, nuc10 and nuc20. Oppositely, nucleosomes with no or fewer SESEs (coloured in orange) consisting of the rest of the +1 nucleosomes, are those with two or fewer SESEs on each strand. Correlation coefficients for each class of nucleosomes under each incubation time are also indicated.

Techniques Used: Sequencing, Incubation

4) Product Images from "MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast"

Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky502

Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.
Figure Legend Snippet: Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.

Techniques Used:

MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.
Figure Legend Snippet: MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.

Techniques Used: Incubation, Atomic Absorption Spectroscopy

Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).
Figure Legend Snippet: Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).

Techniques Used: Sequencing

Correlation between MNase digestions and contents of site-exposure sequence by comparing the two ends of a nucleosome. Correlations between MNase digestions and AA/TT contents in the 1- and 3-min assays are shown on the left and right panels of ( A ), respectively. Similarly, ( B ) shows the correlations of MNase digestion versus AAAA/TTTT content from the 1- and 3-min assays. The shaded rectangle regions in red indicate that the entry site of a nucleosome with more AA/TTs or AAAA/TTTTs gets more digested; the regions in blue indicate that the exit site with more AA/TTs or AAAA/TTTTs gets more digested. The 20 +1 nucleosomes are divided into two groups based on the numbers of s ite e xposure s equence e lements (SESEs, defined as discrete AAAA or TTTT segments) in their sequences. Specifically, nucleosomes with SESEs (coloured in black) are those with three or more SESEs on either strand of the nucleosomes, including nuc01, nuc02, nuc03, nuc05, nuc07, nuc10 and nuc20. Oppositely, nucleosomes with no or fewer SESEs (coloured in orange) consisting of the rest of the +1 nucleosomes, are those with two or fewer SESEs on each strand. Correlation coefficients for each class of nucleosomes under each incubation time are also indicated.
Figure Legend Snippet: Correlation between MNase digestions and contents of site-exposure sequence by comparing the two ends of a nucleosome. Correlations between MNase digestions and AA/TT contents in the 1- and 3-min assays are shown on the left and right panels of ( A ), respectively. Similarly, ( B ) shows the correlations of MNase digestion versus AAAA/TTTT content from the 1- and 3-min assays. The shaded rectangle regions in red indicate that the entry site of a nucleosome with more AA/TTs or AAAA/TTTTs gets more digested; the regions in blue indicate that the exit site with more AA/TTs or AAAA/TTTTs gets more digested. The 20 +1 nucleosomes are divided into two groups based on the numbers of s ite e xposure s equence e lements (SESEs, defined as discrete AAAA or TTTT segments) in their sequences. Specifically, nucleosomes with SESEs (coloured in black) are those with three or more SESEs on either strand of the nucleosomes, including nuc01, nuc02, nuc03, nuc05, nuc07, nuc10 and nuc20. Oppositely, nucleosomes with no or fewer SESEs (coloured in orange) consisting of the rest of the +1 nucleosomes, are those with two or fewer SESEs on each strand. Correlation coefficients for each class of nucleosomes under each incubation time are also indicated.

Techniques Used: Sequencing, Incubation

5) Product Images from "MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast"

Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky502

Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.
Figure Legend Snippet: Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.

Techniques Used:

MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.
Figure Legend Snippet: MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.

Techniques Used: Incubation, Atomic Absorption Spectroscopy

Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).
Figure Legend Snippet: Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).

Techniques Used: Sequencing

6) Product Images from "MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast"

Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky502

Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.
Figure Legend Snippet: Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.

Techniques Used:

MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.
Figure Legend Snippet: MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.

Techniques Used: Incubation, Atomic Absorption Spectroscopy

Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).
Figure Legend Snippet: Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).

Techniques Used: Sequencing

7) Product Images from "Crystal structure and stable property of the cancer-associated heterotypic nucleosome containing CENP-A and H3.3"

Article Title: Crystal structure and stable property of the cancer-associated heterotypic nucleosome containing CENP-A and H3.3

Journal: Scientific Reports

doi: 10.1038/srep07115

The DNA end close to CENP-A is asymmetrically flexible in the CENP-A/H3.3 nucleosome. (a) MNase assay. The H3.3, CENP-A, and CENP-A/H3.3 nucleosomes were treated with MNase (0, 0.3, 0.5 and 0.7 units), and the resulting DNA fragments were analyzed by native PAGE. The gel image shown is a representative of four independent experiments, in which similar results were obtained. (b) ExoIII assay. The H3.3, CENP-A, or CENP-A/H3.3 nucleosomes were incubated with or without 2.0 units of ExoIII for 2.5, 5 and 7.5 minutes at 37°C, and the resultant DNA fragments were extracted and analyzed by 14% denaturing PAGE with 7 M urea. The gel image shown is a representative of three independent experiments, in which similar results were obtained.
Figure Legend Snippet: The DNA end close to CENP-A is asymmetrically flexible in the CENP-A/H3.3 nucleosome. (a) MNase assay. The H3.3, CENP-A, and CENP-A/H3.3 nucleosomes were treated with MNase (0, 0.3, 0.5 and 0.7 units), and the resulting DNA fragments were analyzed by native PAGE. The gel image shown is a representative of four independent experiments, in which similar results were obtained. (b) ExoIII assay. The H3.3, CENP-A, or CENP-A/H3.3 nucleosomes were incubated with or without 2.0 units of ExoIII for 2.5, 5 and 7.5 minutes at 37°C, and the resultant DNA fragments were extracted and analyzed by 14% denaturing PAGE with 7 M urea. The gel image shown is a representative of three independent experiments, in which similar results were obtained.

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

The DNA end close to CENP-A is asymmetrically flexible in the CENP-A/H3.3 nucleosome. (a) MNase assay. The H3.3, CENP-A, and CENP-A/H3.3 nucleosomes were treated with MNase (0, 0.3, 0.5 and 0.7 units), and the resulting DNA fragments were analyzed by native PAGE. The gel image shown is a representative of four independent experiments, in which similar results were obtained. (b) ExoIII assay. The H3.3, CENP-A, or CENP-A/H3.3 nucleosomes were incubated with or without 2.0 units of ExoIII for 2.5, 5 and 7.5 minutes at 37°C, and the resultant DNA fragments were extracted and analyzed by 14% denaturing PAGE with 7 M urea. The gel image shown is a representative of three independent experiments, in which similar results were obtained.
Figure Legend Snippet: The DNA end close to CENP-A is asymmetrically flexible in the CENP-A/H3.3 nucleosome. (a) MNase assay. The H3.3, CENP-A, and CENP-A/H3.3 nucleosomes were treated with MNase (0, 0.3, 0.5 and 0.7 units), and the resulting DNA fragments were analyzed by native PAGE. The gel image shown is a representative of four independent experiments, in which similar results were obtained. (b) ExoIII assay. The H3.3, CENP-A, or CENP-A/H3.3 nucleosomes were incubated with or without 2.0 units of ExoIII for 2.5, 5 and 7.5 minutes at 37°C, and the resultant DNA fragments were extracted and analyzed by 14% denaturing PAGE with 7 M urea. The gel image shown is a representative of three independent experiments, in which similar results were obtained.

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

8) Product Images from "MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast"

Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky502

Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.
Figure Legend Snippet: Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.

Techniques Used:

MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.
Figure Legend Snippet: MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.

Techniques Used: Incubation, Atomic Absorption Spectroscopy

Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).
Figure Legend Snippet: Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).

Techniques Used: Sequencing

Related Articles

Clone Assay:

Article Title: Chickens possess centromeres with both extended tandem repeats and short non-tandem-repetitive sequences
Article Snippet: Paragraph title: Immunoprecipitation, cloning, and sequencing of centromere DNA ... For immnoprecipitation of these cells with an Flag antibody, a nuclear fraction of cells expressing CENPA-Flag was collected and digested with excess MNase (3 U/mL) (Takara) at 37°C for 2 h. The samples were solubilized in 0.5 M NaCl.

Centrifugation:

Article Title: Constitutive centromere-associated network controls centromere drift in vertebrate cells
Article Snippet: ChIP-seq analysis Nuclei were isolated from 1.5 × 109 DT40 cells and digested with 60 units/ml MNase (Takara Bio Inc.) in buffer A (15 mM Hepes-KOH, pH 7.4, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2 , 0.34 M sucrose, 0.5 mM spermidine, 0.15 mM spermine, 1 mM DTT, and 1× complete protease inhibitor cocktail; Roche). .. After centrifugation at 17,800 g for 5 min, the chromatin pellet was suspended with buffer B (20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 10 mM EDTA, and 1× complete protease inhibitor cocktail; Roche), and then mononucleosome was extracted.

Article Title: Interdependency and phosphorylation of KIF4 and condensin I are essential for organization of chromosome scaffold
Article Snippet: .. The chromatin pellets were then re-suspended in CSK2 buffer (10 mM PIPES, pH 6.8, 10% glycerol, 3 mM MgCl2 , 250 mM NaCl, 2.5 mM CaCl2 , 1 mM DTT, 0.25 mM PMSF) containing 20 units MNase (Takara) and incubated at 37°C for 30 min. After centrifugation at 16,100 g for 10 min, supernatant was collected as chromatin-bound protein fraction. .. Note: All CSK buffers contained complete mini protease inhibitor cocktail (Roche) and phosphatase inhibitor PhosSTOP (Roche) and were freshly prepared.

Article Title: Auxin-regulated chromatin switch directs acquisition of flower primordium founder fate
Article Snippet: After centrifugation at 7300 rpm for 5 min, the nuclear fraction was resuspended in HBC buffer ( ). .. The chromatin was digested with a final concentration of 0.02 units/μl MNase (Takara).

Blocking Assay:

Article Title: Interdependency and phosphorylation of KIF4 and condensin I are essential for organization of chromosome scaffold
Article Snippet: Preparation of chromatin-bound or chromatin-unbound protein fractions HeLa or HEK293T cells were synchronized by thymidine block and release in medium containing 100 ng/ml nocodazole. .. The chromatin pellets were then re-suspended in CSK2 buffer (10 mM PIPES, pH 6.8, 10% glycerol, 3 mM MgCl2 , 250 mM NaCl, 2.5 mM CaCl2 , 1 mM DTT, 0.25 mM PMSF) containing 20 units MNase (Takara) and incubated at 37°C for 30 min. After centrifugation at 16,100 g for 10 min, supernatant was collected as chromatin-bound protein fraction.

Incubation:

Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast
Article Snippet: .. The nucleosome mixture (94 nM), containing all types of nucleosomes, was incubated with 5.5 units/ml of MNase (Takara) at 37°C for 1, 3, 6 and 10 min, in 50 mM Tris–HCl (pH 8.0) buffer, containing 2.5 mM CaCl2 , 69 mM NaCl, 81 mM KCl and 1.9 mM dithiothreitol. .. The reaction was stopped by adding half volume of deproteinization solution (20 mM Tris–HCl (pH 8.0), 20 mM EDTA, 0.5 mg/ml proteinase K (Roche) and 0.1% sodium dodecyl sulphate).

Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast
Article Snippet: .. For the MNase digestion experiments with nuc19 and its mutants , each nucleosome (94 nM) was incubated with 5.5 units/ml of MNase (Takara) at 37°C for 1 and 3 min under the same conditions as described above. ..

Article Title: Chickens possess centromeres with both extended tandem repeats and short non-tandem-repetitive sequences
Article Snippet: For immnoprecipitation of these cells with an Flag antibody, a nuclear fraction of cells expressing CENPA-Flag was collected and digested with excess MNase (3 U/mL) (Takara) at 37°C for 2 h. The samples were solubilized in 0.5 M NaCl. .. Anti-Flag M2-beads (Sigma) were incubated with the sample for 2–4 h at 4°C and washed four times with 1 mL of buffer B (20 mM Tris-HCl at pH 8.0, 5 mM EDTA, 500 mM NaCl, and 0.2% Tween 20) and eluted with buffer B in the presence of 3× Flag peptide (Sigma).

Article Title: Constitutive centromere-associated network controls centromere drift in vertebrate cells
Article Snippet: ChIP-seq analysis Nuclei were isolated from 1.5 × 109 DT40 cells and digested with 60 units/ml MNase (Takara Bio Inc.) in buffer A (15 mM Hepes-KOH, pH 7.4, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2 , 0.34 M sucrose, 0.5 mM spermidine, 0.15 mM spermine, 1 mM DTT, and 1× complete protease inhibitor cocktail; Roche). .. The extracted mononucleosome fraction was incubated for 2 h at 4°C with Protein G Sepharose beads (GE Healthcare), which were preincubated with rabbit polyclonal anti–chicken CENP-A antibody ( ).

Article Title: Interdependency and phosphorylation of KIF4 and condensin I are essential for organization of chromosome scaffold
Article Snippet: .. The chromatin pellets were then re-suspended in CSK2 buffer (10 mM PIPES, pH 6.8, 10% glycerol, 3 mM MgCl2 , 250 mM NaCl, 2.5 mM CaCl2 , 1 mM DTT, 0.25 mM PMSF) containing 20 units MNase (Takara) and incubated at 37°C for 30 min. After centrifugation at 16,100 g for 10 min, supernatant was collected as chromatin-bound protein fraction. .. Note: All CSK buffers contained complete mini protease inhibitor cocktail (Roche) and phosphatase inhibitor PhosSTOP (Roche) and were freshly prepared.

Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast
Article Snippet: .. For the MNase digestion experiments with free DNAs, DNA mixture (94 nM) was incubated with 0.5 units/ml of MNase (Takara) at 37°C for 1 and 3 min under the same conditions as nucleosome digestion experiments. .. The DNA fragments were purified by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation.

Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast
Article Snippet: .. MNase digestion assays The nucleosome mixture (94 nM), containing all types of nucleosomes, was incubated with 5.5 units/ml of MNase (Takara) at 37°C for 1, 3, 6 and 10 min, in 50 mM Tris–HCl (pH 8.0) buffer, containing 2.5 mM CaCl2 , 69 mM NaCl, 81 mM KCl and 1.9 mM dithiothreitol. .. The reaction was stopped by adding half volume of deproteinization solution (20 mM Tris–HCl (pH 8.0), 20 mM EDTA, 0.5 mg/ml proteinase K (Roche) and 0.1% sodium dodecyl sulphate).

Article Title: Crystal structure and stable property of the cancer-associated heterotypic nucleosome containing CENP-A and H3.3
Article Snippet: MNase and exonuclease III treatment assays The H3.3, CENP-A, or CENP-A/H3.3 nucleosomes (0.4 μM) were treated with MNase (Takara) or ExoIII (Takara). .. For the MNase assay, the nucleosome samples were incubated with MNase (0, 0.3, 0.5 and 0.7 units) for 5 minutes at 25°C in 10 μl of 44 mM Tris-HCl (pH 8.0) buffer, containing 15 mM NaCl, 2.5 mM CaCl2 , and 1.9 mM dithiothreitol.

Article Title: Promoter Region-Specific Histone Incorporation by the Novel Histone Chaperone ANP32B and DNA-Binding Factor KLF5 ▿
Article Snippet: .. Nuclei were incubated with CaCl2 at a final concentration of 2 mM and 20 units of MNase (Takara) at 37°C for 5 min, and then reactions were stopped by addition of EDTA to a concentration of 10 mM. .. Soluble chromatin was harvested as previously described ( ) and incubated with 8 μg of rabbit polyclonal anti-H4 (H-97; Santa Cruz), whose epitope corresponds to amino acids residues 7 to 103 of the N terminus of human histone H4; rabbit polyclonal anti-H2B (Abcam), whose epitope is derived from a peptide region containing from amino acid residue 100 to the C terminus of human histone H2B; or normal rabbit IgG (Santa Cruz) and then adsorbed to protein G Sepharose (GE Healthcare).

Expressing:

Article Title: Chickens possess centromeres with both extended tandem repeats and short non-tandem-repetitive sequences
Article Snippet: .. For immnoprecipitation of these cells with an Flag antibody, a nuclear fraction of cells expressing CENPA-Flag was collected and digested with excess MNase (3 U/mL) (Takara) at 37°C for 2 h. The samples were solubilized in 0.5 M NaCl. .. Anti-Flag M2-beads (Sigma) were incubated with the sample for 2–4 h at 4°C and washed four times with 1 mL of buffer B (20 mM Tris-HCl at pH 8.0, 5 mM EDTA, 500 mM NaCl, and 0.2% Tween 20) and eluted with buffer B in the presence of 3× Flag peptide (Sigma).

Modification:

Article Title: Auxin-regulated chromatin switch directs acquisition of flower primordium founder fate
Article Snippet: MNase in protoplasts was performed as in intact tissues with some modification. .. The chromatin was digested with a final concentration of 0.02 units/μl MNase (Takara).

Article Title: Auxin decreases chromatin accessibility through the TIR1/AFBs auxin signaling pathway in proliferative cells
Article Snippet: Micrococcal nuclease (MNase) assay After the 2-day treatment to deplete auxin, nuclei were isolated by a modified protocol . .. Digestion was performed by adding 0.5 and 0.25 U/mL MNase (Takara, Shiga, Japan) to the nuclei suspension of BY-2 and MM2d, respectively.

Article Title: Promoter Region-Specific Histone Incorporation by the Novel Histone Chaperone ANP32B and DNA-Binding Factor KLF5 ▿
Article Snippet: Antihistone ChIP assays were performed as previously described ( ) with modification of nucleus isolation according to previously described methods for nucleosome mapping with nuclease ( , ). .. Nuclei were incubated with CaCl2 at a final concentration of 2 mM and 20 units of MNase (Takara) at 37°C for 5 min, and then reactions were stopped by addition of EDTA to a concentration of 10 mM.

Derivative Assay:

Article Title: Auxin decreases chromatin accessibility through the TIR1/AFBs auxin signaling pathway in proliferative cells
Article Snippet: Digestion was performed by adding 0.5 and 0.25 U/mL MNase (Takara, Shiga, Japan) to the nuclei suspension of BY-2 and MM2d, respectively. .. The digested chromatin DNA was electrophoresed on a 1.5% agarose gel and the DNA concentration was determined by measuring the fluorescence derived from ethidium bromide staining using ImageJ.

Article Title: Promoter Region-Specific Histone Incorporation by the Novel Histone Chaperone ANP32B and DNA-Binding Factor KLF5 ▿
Article Snippet: Nuclei were incubated with CaCl2 at a final concentration of 2 mM and 20 units of MNase (Takara) at 37°C for 5 min, and then reactions were stopped by addition of EDTA to a concentration of 10 mM. .. Soluble chromatin was harvested as previously described ( ) and incubated with 8 μg of rabbit polyclonal anti-H4 (H-97; Santa Cruz), whose epitope corresponds to amino acids residues 7 to 103 of the N terminus of human histone H4; rabbit polyclonal anti-H2B (Abcam), whose epitope is derived from a peptide region containing from amino acid residue 100 to the C terminus of human histone H2B; or normal rabbit IgG (Santa Cruz) and then adsorbed to protein G Sepharose (GE Healthcare).

Immunoprecipitation:

Article Title: Chickens possess centromeres with both extended tandem repeats and short non-tandem-repetitive sequences
Article Snippet: Paragraph title: Immunoprecipitation, cloning, and sequencing of centromere DNA ... For immnoprecipitation of these cells with an Flag antibody, a nuclear fraction of cells expressing CENPA-Flag was collected and digested with excess MNase (3 U/mL) (Takara) at 37°C for 2 h. The samples were solubilized in 0.5 M NaCl.

Protease Inhibitor:

Article Title: Constitutive centromere-associated network controls centromere drift in vertebrate cells
Article Snippet: .. ChIP-seq analysis Nuclei were isolated from 1.5 × 109 DT40 cells and digested with 60 units/ml MNase (Takara Bio Inc.) in buffer A (15 mM Hepes-KOH, pH 7.4, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2 , 0.34 M sucrose, 0.5 mM spermidine, 0.15 mM spermine, 1 mM DTT, and 1× complete protease inhibitor cocktail; Roche). .. After centrifugation at 17,800 g for 5 min, the chromatin pellet was suspended with buffer B (20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 10 mM EDTA, and 1× complete protease inhibitor cocktail; Roche), and then mononucleosome was extracted.

Article Title: Interdependency and phosphorylation of KIF4 and condensin I are essential for organization of chromosome scaffold
Article Snippet: The chromatin pellets were then re-suspended in CSK2 buffer (10 mM PIPES, pH 6.8, 10% glycerol, 3 mM MgCl2 , 250 mM NaCl, 2.5 mM CaCl2 , 1 mM DTT, 0.25 mM PMSF) containing 20 units MNase (Takara) and incubated at 37°C for 30 min. After centrifugation at 16,100 g for 10 min, supernatant was collected as chromatin-bound protein fraction. .. Note: All CSK buffers contained complete mini protease inhibitor cocktail (Roche) and phosphatase inhibitor PhosSTOP (Roche) and were freshly prepared.

DNA Sequencing:

Article Title: Chickens possess centromeres with both extended tandem repeats and short non-tandem-repetitive sequences
Article Snippet: For immnoprecipitation of these cells with an Flag antibody, a nuclear fraction of cells expressing CENPA-Flag was collected and digested with excess MNase (3 U/mL) (Takara) at 37°C for 2 h. The samples were solubilized in 0.5 M NaCl. .. Alternatively, the DNA was subjected to Illumina DNA sequencing.

Sequencing:

Article Title: Chickens possess centromeres with both extended tandem repeats and short non-tandem-repetitive sequences
Article Snippet: Paragraph title: Immunoprecipitation, cloning, and sequencing of centromere DNA ... For immnoprecipitation of these cells with an Flag antibody, a nuclear fraction of cells expressing CENPA-Flag was collected and digested with excess MNase (3 U/mL) (Takara) at 37°C for 2 h. The samples were solubilized in 0.5 M NaCl.

ChIP-sequencing:

Article Title: Constitutive centromere-associated network controls centromere drift in vertebrate cells
Article Snippet: .. ChIP-seq analysis Nuclei were isolated from 1.5 × 109 DT40 cells and digested with 60 units/ml MNase (Takara Bio Inc.) in buffer A (15 mM Hepes-KOH, pH 7.4, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2 , 0.34 M sucrose, 0.5 mM spermidine, 0.15 mM spermine, 1 mM DTT, and 1× complete protease inhibitor cocktail; Roche). .. After centrifugation at 17,800 g for 5 min, the chromatin pellet was suspended with buffer B (20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 10 mM EDTA, and 1× complete protease inhibitor cocktail; Roche), and then mononucleosome was extracted.

Fluorescence:

Article Title: Auxin decreases chromatin accessibility through the TIR1/AFBs auxin signaling pathway in proliferative cells
Article Snippet: Digestion was performed by adding 0.5 and 0.25 U/mL MNase (Takara, Shiga, Japan) to the nuclei suspension of BY-2 and MM2d, respectively. .. The digested chromatin DNA was electrophoresed on a 1.5% agarose gel and the DNA concentration was determined by measuring the fluorescence derived from ethidium bromide staining using ImageJ.

Isolation:

Article Title: Constitutive centromere-associated network controls centromere drift in vertebrate cells
Article Snippet: .. ChIP-seq analysis Nuclei were isolated from 1.5 × 109 DT40 cells and digested with 60 units/ml MNase (Takara Bio Inc.) in buffer A (15 mM Hepes-KOH, pH 7.4, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2 , 0.34 M sucrose, 0.5 mM spermidine, 0.15 mM spermine, 1 mM DTT, and 1× complete protease inhibitor cocktail; Roche). .. After centrifugation at 17,800 g for 5 min, the chromatin pellet was suspended with buffer B (20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 10 mM EDTA, and 1× complete protease inhibitor cocktail; Roche), and then mononucleosome was extracted.

Article Title: Auxin decreases chromatin accessibility through the TIR1/AFBs auxin signaling pathway in proliferative cells
Article Snippet: Micrococcal nuclease (MNase) assay After the 2-day treatment to deplete auxin, nuclei were isolated by a modified protocol . .. Digestion was performed by adding 0.5 and 0.25 U/mL MNase (Takara, Shiga, Japan) to the nuclei suspension of BY-2 and MM2d, respectively.

Article Title: Promoter Region-Specific Histone Incorporation by the Novel Histone Chaperone ANP32B and DNA-Binding Factor KLF5 ▿
Article Snippet: Antihistone ChIP assays were performed as previously described ( ) with modification of nucleus isolation according to previously described methods for nucleosome mapping with nuclease ( , ). .. Nuclei were incubated with CaCl2 at a final concentration of 2 mM and 20 units of MNase (Takara) at 37°C for 5 min, and then reactions were stopped by addition of EDTA to a concentration of 10 mM.

Purification:

Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast
Article Snippet: The nucleosome mixture (94 nM), containing all types of nucleosomes, was incubated with 5.5 units/ml of MNase (Takara) at 37°C for 1, 3, 6 and 10 min, in 50 mM Tris–HCl (pH 8.0) buffer, containing 2.5 mM CaCl2 , 69 mM NaCl, 81 mM KCl and 1.9 mM dithiothreitol. .. The DNA fragments were purified by using Wizard SV Gel and PCR Clean-Up System (Promega).

Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast
Article Snippet: The DNA fragments were purified by using Wizard SV Gel and PCR Clean-Up System (Promega). .. For the MNase digestion experiments with nuc19 and its mutants , each nucleosome (94 nM) was incubated with 5.5 units/ml of MNase (Takara) at 37°C for 1 and 3 min under the same conditions as described above.

Article Title: Constitutive centromere-associated network controls centromere drift in vertebrate cells
Article Snippet: ChIP-seq analysis Nuclei were isolated from 1.5 × 109 DT40 cells and digested with 60 units/ml MNase (Takara Bio Inc.) in buffer A (15 mM Hepes-KOH, pH 7.4, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2 , 0.34 M sucrose, 0.5 mM spermidine, 0.15 mM spermine, 1 mM DTT, and 1× complete protease inhibitor cocktail; Roche). .. Beads were washed with buffer B four times, and the bound DNA was purified by phenol-chloroform extraction and ethanol precipitation.

Polymerase Chain Reaction:

Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast
Article Snippet: The nucleosome mixture (94 nM), containing all types of nucleosomes, was incubated with 5.5 units/ml of MNase (Takara) at 37°C for 1, 3, 6 and 10 min, in 50 mM Tris–HCl (pH 8.0) buffer, containing 2.5 mM CaCl2 , 69 mM NaCl, 81 mM KCl and 1.9 mM dithiothreitol. .. The DNA fragments were purified by using Wizard SV Gel and PCR Clean-Up System (Promega).

Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast
Article Snippet: The DNA fragments were purified by using Wizard SV Gel and PCR Clean-Up System (Promega). .. For the MNase digestion experiments with nuc19 and its mutants , each nucleosome (94 nM) was incubated with 5.5 units/ml of MNase (Takara) at 37°C for 1 and 3 min under the same conditions as described above.

Construct:

Article Title: Chickens possess centromeres with both extended tandem repeats and short non-tandem-repetitive sequences
Article Snippet: A CENPA-Flag expression construct under control of the CMV promoter was created. .. For immnoprecipitation of these cells with an Flag antibody, a nuclear fraction of cells expressing CENPA-Flag was collected and digested with excess MNase (3 U/mL) (Takara) at 37°C for 2 h. The samples were solubilized in 0.5 M NaCl.

Lysis:

Article Title: Auxin-regulated chromatin switch directs acquisition of flower primordium founder fate
Article Snippet: 2 × 106 cells were harvest by centrifugation at 11,800 rpm for 2 min, followed by resuspension in 500 µl lysis buffer by vortexing. .. The chromatin was digested with a final concentration of 0.02 units/μl MNase (Takara).

Article Title: Promoter Region-Specific Histone Incorporation by the Novel Histone Chaperone ANP32B and DNA-Binding Factor KLF5 ▿
Article Snippet: Cells were harvested and treated in cell lysis buffer (10 mM Tris HCl [pH 7.5], 10 mM NaCl, 3 mM MgCl2 , 0.5% NP-40, 0.15 mM spermine, 0.5 mM spermidine, and 50 mM sodium butyrate) and centrifuged at 8,000 rpm for 4 min at 4°C, and then precipitate was harvested as nuclei. .. Nuclei were incubated with CaCl2 at a final concentration of 2 mM and 20 units of MNase (Takara) at 37°C for 5 min, and then reactions were stopped by addition of EDTA to a concentration of 10 mM.

Chromatin Immunoprecipitation:

Article Title: Promoter Region-Specific Histone Incorporation by the Novel Histone Chaperone ANP32B and DNA-Binding Factor KLF5 ▿
Article Snippet: Paragraph title: (iii) Anti-histone H2B and H4 ChIP assays. ... Nuclei were incubated with CaCl2 at a final concentration of 2 mM and 20 units of MNase (Takara) at 37°C for 5 min, and then reactions were stopped by addition of EDTA to a concentration of 10 mM.

Agarose Gel Electrophoresis:

Article Title: Histone H1 null vertebrate cells exhibit altered nucleosome architecture
Article Snippet: Nuclei were digested with MNase (Takara) for 5 min at 37°C (0.05 and 0.4 U MNase for 4 × 106 nuclei/20 µl). .. NRL measurement was done in 1% agarose gel in Tris acetate buffer.

Article Title: Auxin decreases chromatin accessibility through the TIR1/AFBs auxin signaling pathway in proliferative cells
Article Snippet: Digestion was performed by adding 0.5 and 0.25 U/mL MNase (Takara, Shiga, Japan) to the nuclei suspension of BY-2 and MM2d, respectively. .. The digested chromatin DNA was electrophoresed on a 1.5% agarose gel and the DNA concentration was determined by measuring the fluorescence derived from ethidium bromide staining using ImageJ.

Ethanol Precipitation:

Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast
Article Snippet: For the MNase digestion experiments with nuc19 and its mutants , each nucleosome (94 nM) was incubated with 5.5 units/ml of MNase (Takara) at 37°C for 1 and 3 min under the same conditions as described above. .. The DNA fragments were purified by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation.

Article Title: Constitutive centromere-associated network controls centromere drift in vertebrate cells
Article Snippet: ChIP-seq analysis Nuclei were isolated from 1.5 × 109 DT40 cells and digested with 60 units/ml MNase (Takara Bio Inc.) in buffer A (15 mM Hepes-KOH, pH 7.4, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2 , 0.34 M sucrose, 0.5 mM spermidine, 0.15 mM spermine, 1 mM DTT, and 1× complete protease inhibitor cocktail; Roche). .. Beads were washed with buffer B four times, and the bound DNA was purified by phenol-chloroform extraction and ethanol precipitation.

Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast
Article Snippet: For the MNase digestion experiments with free DNAs, DNA mixture (94 nM) was incubated with 0.5 units/ml of MNase (Takara) at 37°C for 1 and 3 min under the same conditions as nucleosome digestion experiments. .. The DNA fragments were purified by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation.

Concentration Assay:

Article Title: Auxin-regulated chromatin switch directs acquisition of flower primordium founder fate
Article Snippet: .. The chromatin was digested with a final concentration of 0.02 units/μl MNase (Takara). ..

Article Title: Auxin decreases chromatin accessibility through the TIR1/AFBs auxin signaling pathway in proliferative cells
Article Snippet: Digestion was performed by adding 0.5 and 0.25 U/mL MNase (Takara, Shiga, Japan) to the nuclei suspension of BY-2 and MM2d, respectively. .. The digested chromatin DNA was electrophoresed on a 1.5% agarose gel and the DNA concentration was determined by measuring the fluorescence derived from ethidium bromide staining using ImageJ.

Article Title: Promoter Region-Specific Histone Incorporation by the Novel Histone Chaperone ANP32B and DNA-Binding Factor KLF5 ▿
Article Snippet: .. Nuclei were incubated with CaCl2 at a final concentration of 2 mM and 20 units of MNase (Takara) at 37°C for 5 min, and then reactions were stopped by addition of EDTA to a concentration of 10 mM. .. Soluble chromatin was harvested as previously described ( ) and incubated with 8 μg of rabbit polyclonal anti-H4 (H-97; Santa Cruz), whose epitope corresponds to amino acids residues 7 to 103 of the N terminus of human histone H4; rabbit polyclonal anti-H2B (Abcam), whose epitope is derived from a peptide region containing from amino acid residue 100 to the C terminus of human histone H2B; or normal rabbit IgG (Santa Cruz) and then adsorbed to protein G Sepharose (GE Healthcare).

Staining:

Article Title: Auxin decreases chromatin accessibility through the TIR1/AFBs auxin signaling pathway in proliferative cells
Article Snippet: Digestion was performed by adding 0.5 and 0.25 U/mL MNase (Takara, Shiga, Japan) to the nuclei suspension of BY-2 and MM2d, respectively. .. The digested chromatin DNA was electrophoresed on a 1.5% agarose gel and the DNA concentration was determined by measuring the fluorescence derived from ethidium bromide staining using ImageJ.

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  • 93
    TaKaRa mnase digestion
    Frequencies of occurrence of <t>DNA</t> dinucleotide steps in the +1 nucleosomes of yeast and the sketch of <t>MNase-seq</t> experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.
    Mnase Digestion, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mnase digestion/product/TaKaRa
    Average 93 stars, based on 41 article reviews
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    mnase digestion - by Bioz Stars, 2020-02
    93/100 stars
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    90
    TaKaRa micrococcal nuclease mnase
    Chromatin structure around ade6-3049 is more open than around ade6-3057 , and HRA is involved in the chromatin regulation. (A) <t>MNase</t> sensitivity of chromatin around ade6-3049/3057 . Indicated cells of the pat1-114 background were induced to enter meiosis by temperature-shift method, and harvested 3 hr after the induction. MNase-digested <t>DNA</t> was prepared by MNase (0, 20, or 30 units) treatment of chromatin fraction and subsequent purification. DNA was further cut with Sac I, fractionated on 1.2% agaraose gel, and analyzed by Southern blotting using the probe indicated by the open box. The vertical arrow and the open arrowhead show the ade6 ORF and the 3049/3057 site, respectively. The numbers on the left and the right side of the panel indicate the positions of λ/ Eco T14 I fragments used for electrophoresis marker and the positions from the first A of the ade6 ORF, respectively. The dotted lines indicate a region in which MNase-sensitive sites are clustered around ade6-3049 . Presented is an example from two independent experiments, whose results were similar to each other. (B) HRA deletion reduced acetylation level of H3K9 around ade6-3049 . Indicated cells of the pat1-114 ). DNA isolated from immunoprecipitates and whole-cell extracts was analyzed by real-time qPCR, where fragments corresponding to ade6-3049/3057 and the prp3 + promoter were amplified. Relative enrichment at ade6-3049/3057 over prp3 is shown. Bar graphs are created based on mean values of two independent experiments (shown by ○).
    Micrococcal Nuclease Mnase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/micrococcal nuclease mnase/product/TaKaRa
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    Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.

    Journal: Nucleic Acids Research

    Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast

    doi: 10.1093/nar/gky502

    Figure Lengend Snippet: Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.

    Article Snippet: For the MNase digestion experiments with free DNAs, DNA mixture (94 nM) was incubated with 0.5 units/ml of MNase (Takara) at 37°C for 1 and 3 min under the same conditions as nucleosome digestion experiments.

    Techniques:

    MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.

    Journal: Nucleic Acids Research

    Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast

    doi: 10.1093/nar/gky502

    Figure Lengend Snippet: MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.

    Article Snippet: For the MNase digestion experiments with free DNAs, DNA mixture (94 nM) was incubated with 0.5 units/ml of MNase (Takara) at 37°C for 1 and 3 min under the same conditions as nucleosome digestion experiments.

    Techniques: Incubation, Atomic Absorption Spectroscopy

    Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).

    Journal: Nucleic Acids Research

    Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast

    doi: 10.1093/nar/gky502

    Figure Lengend Snippet: Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).

    Article Snippet: For the MNase digestion experiments with free DNAs, DNA mixture (94 nM) was incubated with 0.5 units/ml of MNase (Takara) at 37°C for 1 and 3 min under the same conditions as nucleosome digestion experiments.

    Techniques: Sequencing

    The proximal region of the DRAM promoter is sensitive to nuclease digestion. (A) Schematic representation of the primer sets used for CHART-PCR. (B) R6 region of the DRAM promoter is more sensitive to DNase I and MNase digestion. The genomic DNA was isolated from Hep3B and HepG2 cells and analyzed by quantitative PCR. The data was shown as percentage of digested DNAs to undigested DNAs. (C) Serum deprivation increases the accessibility of R5, R6 and R7 regions to nuclease digestion in Hep3B and HepG2 cells.

    Journal: PLoS ONE

    Article Title: Serum Starvation Induces DRAM Expression in Liver Cancer Cells via Histone Modifications within Its Promoter Locus

    doi: 10.1371/journal.pone.0050502

    Figure Lengend Snippet: The proximal region of the DRAM promoter is sensitive to nuclease digestion. (A) Schematic representation of the primer sets used for CHART-PCR. (B) R6 region of the DRAM promoter is more sensitive to DNase I and MNase digestion. The genomic DNA was isolated from Hep3B and HepG2 cells and analyzed by quantitative PCR. The data was shown as percentage of digested DNAs to undigested DNAs. (C) Serum deprivation increases the accessibility of R5, R6 and R7 regions to nuclease digestion in Hep3B and HepG2 cells.

    Article Snippet: Chromatin accessibility analysis Accessibility of DNA to DNase I digestion and MNase (Takara, Inc., Dalian, China) were analyzed using chromatin accessibility through CHART-PCR as described previously – .

    Techniques: Polymerase Chain Reaction, Isolation, Real-time Polymerase Chain Reaction

    MNase digestions on nuc19 and its four mutants. Read frequencies of nuc19 and its four mutants from 1-min digestion assays are shown. The digestion products were sequenced via paired-end sequencing approach. In ‘mut_1’ and ‘mut_2’, positions 0–15 on the ‘+’ strand of nuc19 were replaced with the TA-repeated and imperfect poly-A sequences, respectively. In ‘mut_3’ and ‘mut_4’, positions 14–29 on the ‘+’ strand of nuc19 were replaced with the TA-repeated and imperfect poly-A sequences, respectively. The mutated regions are shaded in blue. The frequencies of reads starting from each position in the range from 0 to 40 on the ‘+’ strand are shown in the upper panels. The frequencies of reads terminating at each position on the opposite strand (‘−’) of the mutated regions are shown in the lower panels. Note that frequencies at positions 0(+) and the paired positions on the opposite strand (‘−’) of nuc19, mut_2, mut_3 and mut_4 are above 0.08 and therefore not shown.

    Journal: Nucleic Acids Research

    Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast

    doi: 10.1093/nar/gky502

    Figure Lengend Snippet: MNase digestions on nuc19 and its four mutants. Read frequencies of nuc19 and its four mutants from 1-min digestion assays are shown. The digestion products were sequenced via paired-end sequencing approach. In ‘mut_1’ and ‘mut_2’, positions 0–15 on the ‘+’ strand of nuc19 were replaced with the TA-repeated and imperfect poly-A sequences, respectively. In ‘mut_3’ and ‘mut_4’, positions 14–29 on the ‘+’ strand of nuc19 were replaced with the TA-repeated and imperfect poly-A sequences, respectively. The mutated regions are shaded in blue. The frequencies of reads starting from each position in the range from 0 to 40 on the ‘+’ strand are shown in the upper panels. The frequencies of reads terminating at each position on the opposite strand (‘−’) of the mutated regions are shown in the lower panels. Note that frequencies at positions 0(+) and the paired positions on the opposite strand (‘−’) of nuc19, mut_2, mut_3 and mut_4 are above 0.08 and therefore not shown.

    Article Snippet: For the MNase digestion experiments with nuc19 and its mutants , each nucleosome (94 nM) was incubated with 5.5 units/ml of MNase (Takara) at 37°C for 1 and 3 min under the same conditions as described above.

    Techniques: Sequencing

    Chromatin structure around ade6-3049 is more open than around ade6-3057 , and HRA is involved in the chromatin regulation. (A) MNase sensitivity of chromatin around ade6-3049/3057 . Indicated cells of the pat1-114 background were induced to enter meiosis by temperature-shift method, and harvested 3 hr after the induction. MNase-digested DNA was prepared by MNase (0, 20, or 30 units) treatment of chromatin fraction and subsequent purification. DNA was further cut with Sac I, fractionated on 1.2% agaraose gel, and analyzed by Southern blotting using the probe indicated by the open box. The vertical arrow and the open arrowhead show the ade6 ORF and the 3049/3057 site, respectively. The numbers on the left and the right side of the panel indicate the positions of λ/ Eco T14 I fragments used for electrophoresis marker and the positions from the first A of the ade6 ORF, respectively. The dotted lines indicate a region in which MNase-sensitive sites are clustered around ade6-3049 . Presented is an example from two independent experiments, whose results were similar to each other. (B) HRA deletion reduced acetylation level of H3K9 around ade6-3049 . Indicated cells of the pat1-114 ). DNA isolated from immunoprecipitates and whole-cell extracts was analyzed by real-time qPCR, where fragments corresponding to ade6-3049/3057 and the prp3 + promoter were amplified. Relative enrichment at ade6-3049/3057 over prp3 is shown. Bar graphs are created based on mean values of two independent experiments (shown by ○).

    Journal: Genetics

    Article Title: Correlation of Meiotic DSB Formation and Transcription Initiation Around Fission Yeast Recombination Hotspots

    doi: 10.1534/genetics.116.197954

    Figure Lengend Snippet: Chromatin structure around ade6-3049 is more open than around ade6-3057 , and HRA is involved in the chromatin regulation. (A) MNase sensitivity of chromatin around ade6-3049/3057 . Indicated cells of the pat1-114 background were induced to enter meiosis by temperature-shift method, and harvested 3 hr after the induction. MNase-digested DNA was prepared by MNase (0, 20, or 30 units) treatment of chromatin fraction and subsequent purification. DNA was further cut with Sac I, fractionated on 1.2% agaraose gel, and analyzed by Southern blotting using the probe indicated by the open box. The vertical arrow and the open arrowhead show the ade6 ORF and the 3049/3057 site, respectively. The numbers on the left and the right side of the panel indicate the positions of λ/ Eco T14 I fragments used for electrophoresis marker and the positions from the first A of the ade6 ORF, respectively. The dotted lines indicate a region in which MNase-sensitive sites are clustered around ade6-3049 . Presented is an example from two independent experiments, whose results were similar to each other. (B) HRA deletion reduced acetylation level of H3K9 around ade6-3049 . Indicated cells of the pat1-114 ). DNA isolated from immunoprecipitates and whole-cell extracts was analyzed by real-time qPCR, where fragments corresponding to ade6-3049/3057 and the prp3 + promoter were amplified. Relative enrichment at ade6-3049/3057 over prp3 is shown. Bar graphs are created based on mean values of two independent experiments (shown by ○).

    Article Snippet: Genomic DNA was treated with 0, 20, or 30 units of micrococcal nuclease (MNase) (Takara), digested with Sac I, and separated on a 1.2% agarose gel.

    Techniques: Purification, Southern Blot, Electrophoresis, Marker, Isolation, Real-time Polymerase Chain Reaction, Amplification