Structured Review

Roche mnase
SAMOSA captures bulk and single-molecule evidence of transcription factor-DNA interaction simultaneously via two orthogonal molecular signals. A.-F.) SAMOSA <t>MNase-cut</t> signal averaged over predicted CTCF, NRF1, REST, PU.1, c-MYC, and GATA1 binding motifs in the <t>K562</t> epigenome. All binding sites were predicted from ENCODE ChIP-seq data. G-L.) m 6 dA signal for the same transcription factors, averaged over molecules containing predicted binding sites and at least 250 bases flanking DNA on either side of the predicted motif. Methylation patterns at predicted sites were compared against average profiles taken from randomly drawn molecules from GC%- and repeat-content-matched regions of the genome (calculated for each ENCODE ChIP-seq peak set). M.) Results of clustering motif-containing molecules using the Leiden community detection algorithm. Clusters were manually annotated as containing molecules that were: ‘methylation resistant’ (MR), nucleosome occupied (NO1-8), stochastically accessible (SA1-2), accessible (A), or hyper-accessible (HA). N.) Heatmap representation of single-molecule accessibility profiles for clusters NO7, NO8, and A (500 randomly sampled molecules per cluster). O.) Our data may be explained by the Widom ‘site exposure’ model in vivo . Transcription factor binding motifs are stochastically exposed as nucleosomes toggle between multiple ‘registers’ as seen in Figure 3M (states NO and SA). Transcription factor binding perhaps enforces a favorable nucleosome register (state A), which can then seed hyper-accessible states / further TF-DNA interactions (state HA).
Mnase, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mnase/product/Roche
Average 93 stars, based on 13 article reviews
Price from $9.99 to $1999.99
mnase - by Bioz Stars, 2020-07
93/100 stars

Images

1) Product Images from "Massively multiplex single-molecule oligonucleosome footprinting"

Article Title: Massively multiplex single-molecule oligonucleosome footprinting

Journal: bioRxiv

doi: 10.1101/2020.05.20.105379

SAMOSA captures bulk and single-molecule evidence of transcription factor-DNA interaction simultaneously via two orthogonal molecular signals. A.-F.) SAMOSA MNase-cut signal averaged over predicted CTCF, NRF1, REST, PU.1, c-MYC, and GATA1 binding motifs in the K562 epigenome. All binding sites were predicted from ENCODE ChIP-seq data. G-L.) m 6 dA signal for the same transcription factors, averaged over molecules containing predicted binding sites and at least 250 bases flanking DNA on either side of the predicted motif. Methylation patterns at predicted sites were compared against average profiles taken from randomly drawn molecules from GC%- and repeat-content-matched regions of the genome (calculated for each ENCODE ChIP-seq peak set). M.) Results of clustering motif-containing molecules using the Leiden community detection algorithm. Clusters were manually annotated as containing molecules that were: ‘methylation resistant’ (MR), nucleosome occupied (NO1-8), stochastically accessible (SA1-2), accessible (A), or hyper-accessible (HA). N.) Heatmap representation of single-molecule accessibility profiles for clusters NO7, NO8, and A (500 randomly sampled molecules per cluster). O.) Our data may be explained by the Widom ‘site exposure’ model in vivo . Transcription factor binding motifs are stochastically exposed as nucleosomes toggle between multiple ‘registers’ as seen in Figure 3M (states NO and SA). Transcription factor binding perhaps enforces a favorable nucleosome register (state A), which can then seed hyper-accessible states / further TF-DNA interactions (state HA).
Figure Legend Snippet: SAMOSA captures bulk and single-molecule evidence of transcription factor-DNA interaction simultaneously via two orthogonal molecular signals. A.-F.) SAMOSA MNase-cut signal averaged over predicted CTCF, NRF1, REST, PU.1, c-MYC, and GATA1 binding motifs in the K562 epigenome. All binding sites were predicted from ENCODE ChIP-seq data. G-L.) m 6 dA signal for the same transcription factors, averaged over molecules containing predicted binding sites and at least 250 bases flanking DNA on either side of the predicted motif. Methylation patterns at predicted sites were compared against average profiles taken from randomly drawn molecules from GC%- and repeat-content-matched regions of the genome (calculated for each ENCODE ChIP-seq peak set). M.) Results of clustering motif-containing molecules using the Leiden community detection algorithm. Clusters were manually annotated as containing molecules that were: ‘methylation resistant’ (MR), nucleosome occupied (NO1-8), stochastically accessible (SA1-2), accessible (A), or hyper-accessible (HA). N.) Heatmap representation of single-molecule accessibility profiles for clusters NO7, NO8, and A (500 randomly sampled molecules per cluster). O.) Our data may be explained by the Widom ‘site exposure’ model in vivo . Transcription factor binding motifs are stochastically exposed as nucleosomes toggle between multiple ‘registers’ as seen in Figure 3M (states NO and SA). Transcription factor binding perhaps enforces a favorable nucleosome register (state A), which can then seed hyper-accessible states / further TF-DNA interactions (state HA).

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Methylation, In Vivo

In vivo SAMOSA captures oligonucleosome structure by combining MNase digestion of chromatin with adenine methylation footprinting. A.) An overview of the in vivo SAMOSA protocol: oligonucleosomes are gently solubilized from nuclei using micrococcal nuclease and fusogenic lipid treatment. Resulting oligonucleosomes are footprinted using the EcoGII enzyme and sequencing on the PacBio platform. Each sequencing molecules captures two orthogonal biological signals: MNase cuts that capture ‘barrier’ protein-DNA interactions, and m 6 dA methylation protein-DNA footprints. B.) Fragment length distributions for in vivo SAMOSA data reveal expected oligonucleosomal laddering (bin size = 5 bp). C.) Averaged modification probabilities from SAMOSA experiments demonstrate the ability to mark nucleosome-DNA interactions directly via methylation. Modification patterns seen in the chromatin sample are not seen in unmethylated oligonucleosomal DNA or fully methylated K562 oligonucleosomal DNA.
Figure Legend Snippet: In vivo SAMOSA captures oligonucleosome structure by combining MNase digestion of chromatin with adenine methylation footprinting. A.) An overview of the in vivo SAMOSA protocol: oligonucleosomes are gently solubilized from nuclei using micrococcal nuclease and fusogenic lipid treatment. Resulting oligonucleosomes are footprinted using the EcoGII enzyme and sequencing on the PacBio platform. Each sequencing molecules captures two orthogonal biological signals: MNase cuts that capture ‘barrier’ protein-DNA interactions, and m 6 dA methylation protein-DNA footprints. B.) Fragment length distributions for in vivo SAMOSA data reveal expected oligonucleosomal laddering (bin size = 5 bp). C.) Averaged modification probabilities from SAMOSA experiments demonstrate the ability to mark nucleosome-DNA interactions directly via methylation. Modification patterns seen in the chromatin sample are not seen in unmethylated oligonucleosomal DNA or fully methylated K562 oligonucleosomal DNA.

Techniques Used: In Vivo, Methylation, Footprinting, Sequencing, Modification

2) Product Images from "CENP-A, -B, and -C Chromatin Complex That Contains the I-Type ?-Satellite Array Constitutes the Prekinetochore in HeLa Cells"

Article Title: CENP-A, -B, and -C Chromatin Complex That Contains the I-Type ?-Satellite Array Constitutes the Prekinetochore in HeLa Cells

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.22.7.2229-2241.2002

Bulk chromatin and centromeric chromatin were solubilized by MNase digestion of HeLa nuclei in 0.3 M NaCl. (A) Centromeric proteins CENP-A, -B, and -C were solubilized by MNase digestion. Isolated HeLa nuclei (2 × 10 8 ) were suspended with 1 ml of WB containing 0.3 M NaCl (sample a in lane 1 and sample c in lanes 4 to 6) or 0.6 M NaCl (sample b in lanes 2 and 3). Sample c was digested with 60 U of MNase per ml for 10 min at 37°C. Soluble and insoluble materials from each sample were separated by centrifugation. ACA beads were added to the supernatant of sample c and incubated overnight at 4°C. Pellets were resuspended in 1 ml of SDS buffer by extensive sonication and 5 μl of each sample was separated by SDS-7.5% (for CENP-B and CENP-C) or 12.5% (for CENP-A) PAGE, and centromeric proteins were detected by immunostaining with ACA serum. Lane 1, supernatant fraction of a; lane 2, supernatant fraction of b; lane 3, pellet fraction of b; lane 4, supernatant fraction of c before addition of ACA beads; lane 5, supernatant fraction of c after addition of ACA beads; lane 6, pellet fraction of c. Lane M, marker centromeric proteins, CENP-A, CENP-B, and CENP-C. (B) Size distribution of DNA fragments from bulk chromatin after MNase digestion. HeLa nuclei were digested with MNase to various extents. The fragmented DNA in the soluble fractions was extracted with phenol and electrophoresed through 1% agarose gel. DNA was detected with ethidium bromide staining. Lane 1, 20 U/ml for 2 min (40 U/ml × min, sample 1); lane 2, 20 U/ml for 4 min (80 U/ml × min, sample 2); lane 3, 40 U/ml for 5 min (200 U/ml × min, sample 3); lane 4, 80 U/ml for 45 min (3,600 U/ml × min, sample 4). Positions of the DNA size markers are indicated at the left. (C) Detection of core histones and CENP-A in each fraction. Soluble (sup.) and insoluble (pellet) fractions were subjected to SDS-12.5% PAGE, and the separated core histones were stained with Coomassie brilliant blue (upper panel). The proteins were transferred to a membrane and immunolabeled with ACA serum (AK) (lower panel). Lane M in the lower panel is a recombinant CENP-A marker protein. Lanes 1 to 4 correspond to samples 1 to 4 of the soluble (sup.) fractions, and lanes 5 to 8 to samples 1 to 4 of the pellet fractions.
Figure Legend Snippet: Bulk chromatin and centromeric chromatin were solubilized by MNase digestion of HeLa nuclei in 0.3 M NaCl. (A) Centromeric proteins CENP-A, -B, and -C were solubilized by MNase digestion. Isolated HeLa nuclei (2 × 10 8 ) were suspended with 1 ml of WB containing 0.3 M NaCl (sample a in lane 1 and sample c in lanes 4 to 6) or 0.6 M NaCl (sample b in lanes 2 and 3). Sample c was digested with 60 U of MNase per ml for 10 min at 37°C. Soluble and insoluble materials from each sample were separated by centrifugation. ACA beads were added to the supernatant of sample c and incubated overnight at 4°C. Pellets were resuspended in 1 ml of SDS buffer by extensive sonication and 5 μl of each sample was separated by SDS-7.5% (for CENP-B and CENP-C) or 12.5% (for CENP-A) PAGE, and centromeric proteins were detected by immunostaining with ACA serum. Lane 1, supernatant fraction of a; lane 2, supernatant fraction of b; lane 3, pellet fraction of b; lane 4, supernatant fraction of c before addition of ACA beads; lane 5, supernatant fraction of c after addition of ACA beads; lane 6, pellet fraction of c. Lane M, marker centromeric proteins, CENP-A, CENP-B, and CENP-C. (B) Size distribution of DNA fragments from bulk chromatin after MNase digestion. HeLa nuclei were digested with MNase to various extents. The fragmented DNA in the soluble fractions was extracted with phenol and electrophoresed through 1% agarose gel. DNA was detected with ethidium bromide staining. Lane 1, 20 U/ml for 2 min (40 U/ml × min, sample 1); lane 2, 20 U/ml for 4 min (80 U/ml × min, sample 2); lane 3, 40 U/ml for 5 min (200 U/ml × min, sample 3); lane 4, 80 U/ml for 45 min (3,600 U/ml × min, sample 4). Positions of the DNA size markers are indicated at the left. (C) Detection of core histones and CENP-A in each fraction. Soluble (sup.) and insoluble (pellet) fractions were subjected to SDS-12.5% PAGE, and the separated core histones were stained with Coomassie brilliant blue (upper panel). The proteins were transferred to a membrane and immunolabeled with ACA serum (AK) (lower panel). Lane M in the lower panel is a recombinant CENP-A marker protein. Lanes 1 to 4 correspond to samples 1 to 4 of the soluble (sup.) fractions, and lanes 5 to 8 to samples 1 to 4 of the pellet fractions.

Techniques Used: Isolation, Western Blot, Centrifugation, Incubation, Sonication, Polyacrylamide Gel Electrophoresis, Immunostaining, Marker, Agarose Gel Electrophoresis, Staining, Immunolabeling, Recombinant

3) Product Images from "Maturation-Associated Destabilization of Hepatitis B Virus Nucleocapsid"

Article Title: Maturation-Associated Destabilization of Hepatitis B Virus Nucleocapsid

Journal: Journal of Virology

doi: 10.1128/JVI.01912-13

Nuclease and PK digestion of fractionated and unfractionated NCs. (A) Sucrose gradient fractions 6, 9, and 12 prepared from lysates without prior MNase treatment () were digested with MNase or DNase I as described in Materials and Methods. The reaction
Figure Legend Snippet: Nuclease and PK digestion of fractionated and unfractionated NCs. (A) Sucrose gradient fractions 6, 9, and 12 prepared from lysates without prior MNase treatment () were digested with MNase or DNase I as described in Materials and Methods. The reaction

Techniques Used:

4) Product Images from "Nucleosome positioning at the replication fork"

Article Title: Nucleosome positioning at the replication fork

Journal: The EMBO Journal

doi: 10.1093/emboj/20.24.7294

Fig. 1. Mapping of nucleosome positions in the ribosomal intergenic spacer by the psoralen–exonuclease assay. ( A ) Schematic representation of the method. Crosses represent sites of psoralen cross-links in duplex DNA corresponding to the linker regions between nucleosomes (the preferential sites of psoralen cross-linking). ( B ) Structural organization of the spacer region. Restriction sites (upright and downright arrows), functional elements (boxes) and the transcription start site (+1) are indicated. A Bst EII– Ava II fragment used as a probe for indirect end-labelling is shown (striped box). ( C ) Positioned nucleosomes (numbered boxes 1–5) and the corresponding linkers (roman numerals) are indicated. Positions of the linkers are given with respect to the transcriptional start site. The grey box represents the ORC binding site and adjacent sequences. The large empty box denotes randomly distributed nucleosomes towards the 3′ end of the rDNA coding unit. ( D ) Nucleosome mapping between the 5S gene and the transcription start site by indirect end-labelling from the Bst EII site close to the 5S gene. In the left panel, the untreated Bst EII fragment (N, lane 1), MNase cutting sites in chromatin (C, lanes 3 and 4) or in deproteinized DNA (D, lane 2) are shown. Chromatin (lanes C) was digested for 10 min with 25 U/µl (lane 3) or 500 U/µl (lane 4) MNase. In the right panel, psoralen cross-linking sites in chromatin (C, lanes 5 and 6) and deproteinized DNA (D, lane 7) were determined by the psoralen–exonuclease assay. λ-exonuclease entry sites were created by digesting purified cross-linked DNA for 2 min with 2 U/µl (lane 6) or 10 U/µl (lane 5) MNase [see (A)]. The pBR328 DNA fragments ( Bgl I– Hin fI) used as size markers are shown.
Figure Legend Snippet: Fig. 1. Mapping of nucleosome positions in the ribosomal intergenic spacer by the psoralen–exonuclease assay. ( A ) Schematic representation of the method. Crosses represent sites of psoralen cross-links in duplex DNA corresponding to the linker regions between nucleosomes (the preferential sites of psoralen cross-linking). ( B ) Structural organization of the spacer region. Restriction sites (upright and downright arrows), functional elements (boxes) and the transcription start site (+1) are indicated. A Bst EII– Ava II fragment used as a probe for indirect end-labelling is shown (striped box). ( C ) Positioned nucleosomes (numbered boxes 1–5) and the corresponding linkers (roman numerals) are indicated. Positions of the linkers are given with respect to the transcriptional start site. The grey box represents the ORC binding site and adjacent sequences. The large empty box denotes randomly distributed nucleosomes towards the 3′ end of the rDNA coding unit. ( D ) Nucleosome mapping between the 5S gene and the transcription start site by indirect end-labelling from the Bst EII site close to the 5S gene. In the left panel, the untreated Bst EII fragment (N, lane 1), MNase cutting sites in chromatin (C, lanes 3 and 4) or in deproteinized DNA (D, lane 2) are shown. Chromatin (lanes C) was digested for 10 min with 25 U/µl (lane 3) or 500 U/µl (lane 4) MNase. In the right panel, psoralen cross-linking sites in chromatin (C, lanes 5 and 6) and deproteinized DNA (D, lane 7) were determined by the psoralen–exonuclease assay. λ-exonuclease entry sites were created by digesting purified cross-linked DNA for 2 min with 2 U/µl (lane 6) or 10 U/µl (lane 5) MNase [see (A)]. The pBR328 DNA fragments ( Bgl I– Hin fI) used as size markers are shown.

Techniques Used: Functional Assay, Antiviral Assay, Binding Assay, Purification

5) Product Images from "T helper type 1-specific Brg1 recruitment and remodeling of nucleosomes positioned at the IFN-? promoter are Stat4 dependent"

Article Title: T helper type 1-specific Brg1 recruitment and remodeling of nucleosomes positioned at the IFN-? promoter are Stat4 dependent

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20060066

Nucleosomes positioned in IFN-γ promoter chromatin in CD4 T cells. (a and b) Naive CD4 T cells were cross-linked with formaldehyde, and their chromatin was treated with MNase ( 5 , 7 .5, and 10 enzyme units). DNAs were then purified, digested with the indicated RE (or left undigested; 0), and analyzed by Southern blots using a probe indicated in panel c (directly adjacent to the RE site for HincII-cut DNAs [a] or EcoRI digests [b]). (c) Diagram of RE sites and inferred nucleosome positions at the IFN-γ promoter. The fragment used as a probe in Southern blotting and primers used for LM-PCR are shown above the gene; the transcription start site (+1) is indicated by an arrow. (d) LM-PCR mapping of nucleosome boundaries in the IFN-γ promoter. DNA purified from MNase (2.5 and 1 enzyme unit)-cleaved chromatin of naive or Th1 (6-d culture) CD4 T cells was analyzed by LM-PCR using primer x (nucleosome 1) or y (nucleosome 2) and a linker primer and was analyzed by Southern blot probed with an internal oligonucleotide (1 and 2 for nucleosomes 1 and 2). As a control for MNase cleavage preferences in DNA, pure cellular DNA was analyzed using the same preparation of MNase, linker ligation, and PCR (naked DNA). Shown is an autoradiograph representative of five independent experiments.
Figure Legend Snippet: Nucleosomes positioned in IFN-γ promoter chromatin in CD4 T cells. (a and b) Naive CD4 T cells were cross-linked with formaldehyde, and their chromatin was treated with MNase ( 5 , 7 .5, and 10 enzyme units). DNAs were then purified, digested with the indicated RE (or left undigested; 0), and analyzed by Southern blots using a probe indicated in panel c (directly adjacent to the RE site for HincII-cut DNAs [a] or EcoRI digests [b]). (c) Diagram of RE sites and inferred nucleosome positions at the IFN-γ promoter. The fragment used as a probe in Southern blotting and primers used for LM-PCR are shown above the gene; the transcription start site (+1) is indicated by an arrow. (d) LM-PCR mapping of nucleosome boundaries in the IFN-γ promoter. DNA purified from MNase (2.5 and 1 enzyme unit)-cleaved chromatin of naive or Th1 (6-d culture) CD4 T cells was analyzed by LM-PCR using primer x (nucleosome 1) or y (nucleosome 2) and a linker primer and was analyzed by Southern blot probed with an internal oligonucleotide (1 and 2 for nucleosomes 1 and 2). As a control for MNase cleavage preferences in DNA, pure cellular DNA was analyzed using the same preparation of MNase, linker ligation, and PCR (naked DNA). Shown is an autoradiograph representative of five independent experiments.

Techniques Used: Purification, Southern Blot, Polymerase Chain Reaction, Ligation, Autoradiography

6) Product Images from "The octamer is the major form of CENP-A nucleosomes at human centromeres"

Article Title: The octamer is the major form of CENP-A nucleosomes at human centromeres

Journal: Nature structural & molecular biology

doi: 10.1038/nsmb.2562

Terminally unwrapped CENP-A nucleosomes and their conventional counterparts with wrapped termini are similarly phased at normal centromeres ( a,b ) The position of each individual CENP-A ( a ) or bulk nucleosome ( b ) along a dimerized α-satellite consensus sequence is indicated by a horizontal line. Each fragment is color-coded based on length, as indicated. ( c,d ) The midpoint positions of CENP-A ( c ) or bulk nucleosome ( d ) fragments along the dimer α-satellite consensus sequence. Solid vertical lines indicate the location of the 17 bp CENP-B box ( B ) in ( a–d ). ( e,f ) Models of the preferred positioning and MNase cleavage sites on CENP-A ( e ) and bulk ( f ) nucleosomes at normal centromeres.
Figure Legend Snippet: Terminally unwrapped CENP-A nucleosomes and their conventional counterparts with wrapped termini are similarly phased at normal centromeres ( a,b ) The position of each individual CENP-A ( a ) or bulk nucleosome ( b ) along a dimerized α-satellite consensus sequence is indicated by a horizontal line. Each fragment is color-coded based on length, as indicated. ( c,d ) The midpoint positions of CENP-A ( c ) or bulk nucleosome ( d ) fragments along the dimer α-satellite consensus sequence. Solid vertical lines indicate the location of the 17 bp CENP-B box ( B ) in ( a–d ). ( e,f ) Models of the preferred positioning and MNase cleavage sites on CENP-A ( e ) and bulk ( f ) nucleosomes at normal centromeres.

Techniques Used: Sequencing

CENP-A nucleosomes are less phased and gain symmetric MNase digestion on the Y chromosome centromere that lacks functional CENP-B boxes ( a–c ) Maps of chromosome X HOR-aligned CENP-A sequences. ( d–f ). Maps of chromosome Y HOR-aligned CENP-A sequences. Data and models are shown in the same manner as for the global analysis of CENP-A nucleosome-associated α-satellite sequences ( Fig. 5 ).
Figure Legend Snippet: CENP-A nucleosomes are less phased and gain symmetric MNase digestion on the Y chromosome centromere that lacks functional CENP-B boxes ( a–c ) Maps of chromosome X HOR-aligned CENP-A sequences. ( d–f ). Maps of chromosome Y HOR-aligned CENP-A sequences. Data and models are shown in the same manner as for the global analysis of CENP-A nucleosome-associated α-satellite sequences ( Fig. 5 ).

Techniques Used: Functional Assay

Nuclease digestion of native CENP-A-containing particles resembles that of octameric nucleosomes with loose termini ( a ) DNA length distributions of MNase-digested CENP-A native ChIP and bulk nucleosomes from the same preparation. ( b ) Fluorescence in situ hybridization using DNA from bulk nucleosomes or CENP-A native ChIP as probes. Bulk nucleosome DNA labels the entire chromosome whereas CENP-A probe labels solely centromeric regions, as expected. ( c ) Quantitative real-time PCR analysis comparing enrichment of CENP-A native ChIP DNA relative to bulk nucleosome DNA. CENP-A ChIP sequences are enriched for α-satellite regions (α-satellite1, α-satellite2), but not at pericentric or promoter (aldo) regions, as expected. Error bars represent s.e.m. from three independent replicates. ( d ) Standard digestion (red) or overdigestion (blue, threefold higher concentration of MNase used) of chromatin. ( e ) DNA length distributions of CENP-A native ChIP following standard digestion (red) or overdigestion (blue) of chromatin.
Figure Legend Snippet: Nuclease digestion of native CENP-A-containing particles resembles that of octameric nucleosomes with loose termini ( a ) DNA length distributions of MNase-digested CENP-A native ChIP and bulk nucleosomes from the same preparation. ( b ) Fluorescence in situ hybridization using DNA from bulk nucleosomes or CENP-A native ChIP as probes. Bulk nucleosome DNA labels the entire chromosome whereas CENP-A probe labels solely centromeric regions, as expected. ( c ) Quantitative real-time PCR analysis comparing enrichment of CENP-A native ChIP DNA relative to bulk nucleosome DNA. CENP-A ChIP sequences are enriched for α-satellite regions (α-satellite1, α-satellite2), but not at pericentric or promoter (aldo) regions, as expected. Error bars represent s.e.m. from three independent replicates. ( d ) Standard digestion (red) or overdigestion (blue, threefold higher concentration of MNase used) of chromatin. ( e ) DNA length distributions of CENP-A native ChIP following standard digestion (red) or overdigestion (blue) of chromatin.

Techniques Used: Chromatin Immunoprecipitation, Fluorescence, In Situ Hybridization, Real-time Polymerase Chain Reaction, Concentration Assay

7) Product Images from "CENP-A, -B, and -C Chromatin Complex That Contains the I-Type ?-Satellite Array Constitutes the Prekinetochore in HeLa Cells"

Article Title: CENP-A, -B, and -C Chromatin Complex That Contains the I-Type ?-Satellite Array Constitutes the Prekinetochore in HeLa Cells

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.22.7.2229-2241.2002

Bulk chromatin and centromeric chromatin were solubilized by MNase digestion of HeLa nuclei in 0.3 M NaCl. (A) Centromeric proteins CENP-A, -B, and -C were solubilized by MNase digestion. Isolated HeLa nuclei (2 × 10 8 ) were suspended with 1 ml of WB containing 0.3 M NaCl (sample a in lane 1 and sample c in lanes 4 to 6) or 0.6 M NaCl (sample b in lanes 2 and 3). Sample c was digested with 60 U of MNase per ml for 10 min at 37°C. Soluble and insoluble materials from each sample were separated by centrifugation. ACA beads were added to the supernatant of sample c and incubated overnight at 4°C. Pellets were resuspended in 1 ml of SDS buffer by extensive sonication and 5 μl of each sample was separated by SDS-7.5% (for CENP-B and CENP-C) or 12.5% (for CENP-A) PAGE, and centromeric proteins were detected by immunostaining with ACA serum. Lane 1, supernatant fraction of a; lane 2, supernatant fraction of b; lane 3, pellet fraction of b; lane 4, supernatant fraction of c before addition of ACA beads; lane 5, supernatant fraction of c after addition of ACA beads; lane 6, pellet fraction of c. Lane M, marker centromeric proteins, CENP-A, CENP-B, and CENP-C. (B) Size distribution of DNA fragments from bulk chromatin after MNase digestion. HeLa nuclei were digested with MNase to various extents. The fragmented DNA in the soluble fractions was extracted with phenol and electrophoresed through 1% agarose gel. DNA was detected with ethidium bromide staining. Lane 1, 20 U/ml for 2 min (40 U/ml × min, sample 1); lane 2, 20 U/ml for 4 min (80 U/ml × min, sample 2); lane 3, 40 U/ml for 5 min (200 U/ml × min, sample 3); lane 4, 80 U/ml for 45 min (3,600 U/ml × min, sample 4). Positions of the DNA size markers are indicated at the left. (C) Detection of core histones and CENP-A in each fraction. Soluble (sup.) and insoluble (pellet) fractions were subjected to SDS-12.5% PAGE, and the separated core histones were stained with Coomassie brilliant blue (upper panel). The proteins were transferred to a membrane and immunolabeled with ACA serum (AK) (lower panel). Lane M in the lower panel is a recombinant CENP-A marker protein. Lanes 1 to 4 correspond to samples 1 to 4 of the soluble (sup.) fractions, and lanes 5 to 8 to samples 1 to 4 of the pellet fractions.
Figure Legend Snippet: Bulk chromatin and centromeric chromatin were solubilized by MNase digestion of HeLa nuclei in 0.3 M NaCl. (A) Centromeric proteins CENP-A, -B, and -C were solubilized by MNase digestion. Isolated HeLa nuclei (2 × 10 8 ) were suspended with 1 ml of WB containing 0.3 M NaCl (sample a in lane 1 and sample c in lanes 4 to 6) or 0.6 M NaCl (sample b in lanes 2 and 3). Sample c was digested with 60 U of MNase per ml for 10 min at 37°C. Soluble and insoluble materials from each sample were separated by centrifugation. ACA beads were added to the supernatant of sample c and incubated overnight at 4°C. Pellets were resuspended in 1 ml of SDS buffer by extensive sonication and 5 μl of each sample was separated by SDS-7.5% (for CENP-B and CENP-C) or 12.5% (for CENP-A) PAGE, and centromeric proteins were detected by immunostaining with ACA serum. Lane 1, supernatant fraction of a; lane 2, supernatant fraction of b; lane 3, pellet fraction of b; lane 4, supernatant fraction of c before addition of ACA beads; lane 5, supernatant fraction of c after addition of ACA beads; lane 6, pellet fraction of c. Lane M, marker centromeric proteins, CENP-A, CENP-B, and CENP-C. (B) Size distribution of DNA fragments from bulk chromatin after MNase digestion. HeLa nuclei were digested with MNase to various extents. The fragmented DNA in the soluble fractions was extracted with phenol and electrophoresed through 1% agarose gel. DNA was detected with ethidium bromide staining. Lane 1, 20 U/ml for 2 min (40 U/ml × min, sample 1); lane 2, 20 U/ml for 4 min (80 U/ml × min, sample 2); lane 3, 40 U/ml for 5 min (200 U/ml × min, sample 3); lane 4, 80 U/ml for 45 min (3,600 U/ml × min, sample 4). Positions of the DNA size markers are indicated at the left. (C) Detection of core histones and CENP-A in each fraction. Soluble (sup.) and insoluble (pellet) fractions were subjected to SDS-12.5% PAGE, and the separated core histones were stained with Coomassie brilliant blue (upper panel). The proteins were transferred to a membrane and immunolabeled with ACA serum (AK) (lower panel). Lane M in the lower panel is a recombinant CENP-A marker protein. Lanes 1 to 4 correspond to samples 1 to 4 of the soluble (sup.) fractions, and lanes 5 to 8 to samples 1 to 4 of the pellet fractions.

Techniques Used: Isolation, Western Blot, Centrifugation, Incubation, Sonication, Polyacrylamide Gel Electrophoresis, Immunostaining, Marker, Agarose Gel Electrophoresis, Staining, Immunolabeling, Recombinant

Related Articles

Electron Paramagnetic Resonance:

Article Title: Maturation-Associated Destabilization of Hepatitis B Virus Nucleocapsid
Article Snippet: .. NCs isolated by sucrose gradient centrifugation (10 μl of the indicated sucrose fraction) or cytoplasmic lysate (10 μl) were treated with 1 mg/ml of PK (Invitrogen) (unless noted otherwise) in EPR buffer in a total volume of 20 μl at 37°C for 1 h. Similarly, for nuclease treatment, MNase (0.25 unit/μl) or DNase I (Roche) (2 mg/ml) was added, and the reaction mixture was incubated at 37°C for 1 h. For MNase digestion, the MgCl2 in the EPR buffer was replaced with CaCl2 (5 mM). .. For digestion with both PK and DNase, the samples were first treated with PK for 1 h, followed by the addition of DNase I and incubation for another 1 h. The nucleases were inactivated by the addition of 15 mM EDTA.

Centrifugation:

Article Title: Massively multiplex single-molecule oligonucleosome footprinting
Article Snippet: .. Isolation of nuclei, MNase digest, and overnight dialysis 100E6 K562 cells were collected by centrifugation (300x g , 5 min), washed in ice cold 1X PBS, and resuspended in 1 mL Nuclear Isolation Buffer (20mM HEPES, 10mM KCl, 1mM MgCl2, 0.1% Triton X-100, 20% Glycerol, and 1X Protease Inhibitor (Roche)) per 5-10 e6 cells by gently pipetting 5x with a wide-bore tip to release nuclei. .. The suspension was incubated on ice for 5 minutes, and nuclei were pelleted (600xg, 4°C, 5 min), washed with Buffer M (15mM Tris-HCl pH 8.0, 15 mM NaCl, 60mM KCl, 0.5mM Spermidine), and spun once again.

Protease Inhibitor:

Article Title: Massively multiplex single-molecule oligonucleosome footprinting
Article Snippet: .. Isolation of nuclei, MNase digest, and overnight dialysis 100E6 K562 cells were collected by centrifugation (300x g , 5 min), washed in ice cold 1X PBS, and resuspended in 1 mL Nuclear Isolation Buffer (20mM HEPES, 10mM KCl, 1mM MgCl2, 0.1% Triton X-100, 20% Glycerol, and 1X Protease Inhibitor (Roche)) per 5-10 e6 cells by gently pipetting 5x with a wide-bore tip to release nuclei. .. The suspension was incubated on ice for 5 minutes, and nuclei were pelleted (600xg, 4°C, 5 min), washed with Buffer M (15mM Tris-HCl pH 8.0, 15 mM NaCl, 60mM KCl, 0.5mM Spermidine), and spun once again.

Concentration Assay:

Article Title: CENP-A, -B, and -C Chromatin Complex That Contains the I-Type ?-Satellite Array Constitutes the Prekinetochore in HeLa Cells
Article Snippet: .. The nuclear suspension was digested with MNase (Roche Diagnostics) at 37°C after addition of CaCl2 to a final concentration of 2 mM. .. The reaction was stopped by the addition of EGTA to a final concentration of 5 mM with quick chilling.

Incubation:

Article Title: Nucleosome positioning at the replication fork
Article Snippet: .. Psoralen cross-linked DNA was mildly digested with MNase (or limit digested with an appropriate restriction enzyme) and subsequently incubated for 30 min at 37°C with 5 U of mung bean nuclease (Roche; reaction buffer: 30 mM sodium acetate pH 4.6, 50 mM NaCl, 1 mM zinc acetate, 0.001% Triton X-100). ..

Article Title: Maturation-Associated Destabilization of Hepatitis B Virus Nucleocapsid
Article Snippet: .. NCs isolated by sucrose gradient centrifugation (10 μl of the indicated sucrose fraction) or cytoplasmic lysate (10 μl) were treated with 1 mg/ml of PK (Invitrogen) (unless noted otherwise) in EPR buffer in a total volume of 20 μl at 37°C for 1 h. Similarly, for nuclease treatment, MNase (0.25 unit/μl) or DNase I (Roche) (2 mg/ml) was added, and the reaction mixture was incubated at 37°C for 1 h. For MNase digestion, the MgCl2 in the EPR buffer was replaced with CaCl2 (5 mM). .. For digestion with both PK and DNase, the samples were first treated with PK for 1 h, followed by the addition of DNase I and incubation for another 1 h. The nucleases were inactivated by the addition of 15 mM EDTA.

Isolation:

Article Title: Maturation-Associated Destabilization of Hepatitis B Virus Nucleocapsid
Article Snippet: .. NCs isolated by sucrose gradient centrifugation (10 μl of the indicated sucrose fraction) or cytoplasmic lysate (10 μl) were treated with 1 mg/ml of PK (Invitrogen) (unless noted otherwise) in EPR buffer in a total volume of 20 μl at 37°C for 1 h. Similarly, for nuclease treatment, MNase (0.25 unit/μl) or DNase I (Roche) (2 mg/ml) was added, and the reaction mixture was incubated at 37°C for 1 h. For MNase digestion, the MgCl2 in the EPR buffer was replaced with CaCl2 (5 mM). .. For digestion with both PK and DNase, the samples were first treated with PK for 1 h, followed by the addition of DNase I and incubation for another 1 h. The nucleases were inactivated by the addition of 15 mM EDTA.

Article Title: Massively multiplex single-molecule oligonucleosome footprinting
Article Snippet: .. Isolation of nuclei, MNase digest, and overnight dialysis 100E6 K562 cells were collected by centrifugation (300x g , 5 min), washed in ice cold 1X PBS, and resuspended in 1 mL Nuclear Isolation Buffer (20mM HEPES, 10mM KCl, 1mM MgCl2, 0.1% Triton X-100, 20% Glycerol, and 1X Protease Inhibitor (Roche)) per 5-10 e6 cells by gently pipetting 5x with a wide-bore tip to release nuclei. .. The suspension was incubated on ice for 5 minutes, and nuclei were pelleted (600xg, 4°C, 5 min), washed with Buffer M (15mM Tris-HCl pH 8.0, 15 mM NaCl, 60mM KCl, 0.5mM Spermidine), and spun once again.

Gradient Centrifugation:

Article Title: Maturation-Associated Destabilization of Hepatitis B Virus Nucleocapsid
Article Snippet: .. NCs isolated by sucrose gradient centrifugation (10 μl of the indicated sucrose fraction) or cytoplasmic lysate (10 μl) were treated with 1 mg/ml of PK (Invitrogen) (unless noted otherwise) in EPR buffer in a total volume of 20 μl at 37°C for 1 h. Similarly, for nuclease treatment, MNase (0.25 unit/μl) or DNase I (Roche) (2 mg/ml) was added, and the reaction mixture was incubated at 37°C for 1 h. For MNase digestion, the MgCl2 in the EPR buffer was replaced with CaCl2 (5 mM). .. For digestion with both PK and DNase, the samples were first treated with PK for 1 h, followed by the addition of DNase I and incubation for another 1 h. The nucleases were inactivated by the addition of 15 mM EDTA.

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • mnase  (Roche)
    93
    Roche mnase
    SAMOSA captures bulk and single-molecule evidence of transcription factor-DNA interaction simultaneously via two orthogonal molecular signals. A.-F.) SAMOSA <t>MNase-cut</t> signal averaged over predicted CTCF, NRF1, REST, PU.1, c-MYC, and GATA1 binding motifs in the <t>K562</t> epigenome. All binding sites were predicted from ENCODE ChIP-seq data. G-L.) m 6 dA signal for the same transcription factors, averaged over molecules containing predicted binding sites and at least 250 bases flanking DNA on either side of the predicted motif. Methylation patterns at predicted sites were compared against average profiles taken from randomly drawn molecules from GC%- and repeat-content-matched regions of the genome (calculated for each ENCODE ChIP-seq peak set). M.) Results of clustering motif-containing molecules using the Leiden community detection algorithm. Clusters were manually annotated as containing molecules that were: ‘methylation resistant’ (MR), nucleosome occupied (NO1-8), stochastically accessible (SA1-2), accessible (A), or hyper-accessible (HA). N.) Heatmap representation of single-molecule accessibility profiles for clusters NO7, NO8, and A (500 randomly sampled molecules per cluster). O.) Our data may be explained by the Widom ‘site exposure’ model in vivo . Transcription factor binding motifs are stochastically exposed as nucleosomes toggle between multiple ‘registers’ as seen in Figure 3M (states NO and SA). Transcription factor binding perhaps enforces a favorable nucleosome register (state A), which can then seed hyper-accessible states / further TF-DNA interactions (state HA).
    Mnase, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mnase/product/Roche
    Average 93 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    mnase - by Bioz Stars, 2020-07
    93/100 stars
      Buy from Supplier

    91
    Roche mnase digested dna
    Humanized yeast have trouble adapting to new conditions (A) Violin plots showing that humanized yeast cells are larger and have dysregulated cell size based on phase-contrast microscopy measurements. (B) Cell-cycle ( CLB1 ) and cell-size ( WHI4 ) regulating genes each have highly occupied −1 and −2 nucleosomes. (C) Humanized yeast have a prolonged S-phase and/or arrest in G1. Cell-cycle was analyzed by sytox green straining of <t>DNA</t> content and measured by flow cytometry. Each plot shows 10,000 cells. (D) Humanized yeast have delayed remodeling at the GAL1 promoter. Time-course was analyzed by galactose induction of eGFP using flow cytometry. (E) <t>MNase-seq</t> map of PHO5 promoter, and time-course nucleosome scanning assay using WT, hH3.1-core, and hH3.3-core nucleosome yeasts upon phosphate starvation at different time points. Data points show qPCR amplicon midpoints, and mean ± SD of 2 biological replicates.
    Mnase Digested Dna, supplied by Roche, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mnase digested dna/product/Roche
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mnase digested dna - by Bioz Stars, 2020-07
    91/100 stars
      Buy from Supplier

    Image Search Results


    SAMOSA captures bulk and single-molecule evidence of transcription factor-DNA interaction simultaneously via two orthogonal molecular signals. A.-F.) SAMOSA MNase-cut signal averaged over predicted CTCF, NRF1, REST, PU.1, c-MYC, and GATA1 binding motifs in the K562 epigenome. All binding sites were predicted from ENCODE ChIP-seq data. G-L.) m 6 dA signal for the same transcription factors, averaged over molecules containing predicted binding sites and at least 250 bases flanking DNA on either side of the predicted motif. Methylation patterns at predicted sites were compared against average profiles taken from randomly drawn molecules from GC%- and repeat-content-matched regions of the genome (calculated for each ENCODE ChIP-seq peak set). M.) Results of clustering motif-containing molecules using the Leiden community detection algorithm. Clusters were manually annotated as containing molecules that were: ‘methylation resistant’ (MR), nucleosome occupied (NO1-8), stochastically accessible (SA1-2), accessible (A), or hyper-accessible (HA). N.) Heatmap representation of single-molecule accessibility profiles for clusters NO7, NO8, and A (500 randomly sampled molecules per cluster). O.) Our data may be explained by the Widom ‘site exposure’ model in vivo . Transcription factor binding motifs are stochastically exposed as nucleosomes toggle between multiple ‘registers’ as seen in Figure 3M (states NO and SA). Transcription factor binding perhaps enforces a favorable nucleosome register (state A), which can then seed hyper-accessible states / further TF-DNA interactions (state HA).

    Journal: bioRxiv

    Article Title: Massively multiplex single-molecule oligonucleosome footprinting

    doi: 10.1101/2020.05.20.105379

    Figure Lengend Snippet: SAMOSA captures bulk and single-molecule evidence of transcription factor-DNA interaction simultaneously via two orthogonal molecular signals. A.-F.) SAMOSA MNase-cut signal averaged over predicted CTCF, NRF1, REST, PU.1, c-MYC, and GATA1 binding motifs in the K562 epigenome. All binding sites were predicted from ENCODE ChIP-seq data. G-L.) m 6 dA signal for the same transcription factors, averaged over molecules containing predicted binding sites and at least 250 bases flanking DNA on either side of the predicted motif. Methylation patterns at predicted sites were compared against average profiles taken from randomly drawn molecules from GC%- and repeat-content-matched regions of the genome (calculated for each ENCODE ChIP-seq peak set). M.) Results of clustering motif-containing molecules using the Leiden community detection algorithm. Clusters were manually annotated as containing molecules that were: ‘methylation resistant’ (MR), nucleosome occupied (NO1-8), stochastically accessible (SA1-2), accessible (A), or hyper-accessible (HA). N.) Heatmap representation of single-molecule accessibility profiles for clusters NO7, NO8, and A (500 randomly sampled molecules per cluster). O.) Our data may be explained by the Widom ‘site exposure’ model in vivo . Transcription factor binding motifs are stochastically exposed as nucleosomes toggle between multiple ‘registers’ as seen in Figure 3M (states NO and SA). Transcription factor binding perhaps enforces a favorable nucleosome register (state A), which can then seed hyper-accessible states / further TF-DNA interactions (state HA).

    Article Snippet: Isolation of nuclei, MNase digest, and overnight dialysis 100E6 K562 cells were collected by centrifugation (300x g , 5 min), washed in ice cold 1X PBS, and resuspended in 1 mL Nuclear Isolation Buffer (20mM HEPES, 10mM KCl, 1mM MgCl2, 0.1% Triton X-100, 20% Glycerol, and 1X Protease Inhibitor (Roche)) per 5-10 e6 cells by gently pipetting 5x with a wide-bore tip to release nuclei.

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Methylation, In Vivo

    In vivo SAMOSA captures oligonucleosome structure by combining MNase digestion of chromatin with adenine methylation footprinting. A.) An overview of the in vivo SAMOSA protocol: oligonucleosomes are gently solubilized from nuclei using micrococcal nuclease and fusogenic lipid treatment. Resulting oligonucleosomes are footprinted using the EcoGII enzyme and sequencing on the PacBio platform. Each sequencing molecules captures two orthogonal biological signals: MNase cuts that capture ‘barrier’ protein-DNA interactions, and m 6 dA methylation protein-DNA footprints. B.) Fragment length distributions for in vivo SAMOSA data reveal expected oligonucleosomal laddering (bin size = 5 bp). C.) Averaged modification probabilities from SAMOSA experiments demonstrate the ability to mark nucleosome-DNA interactions directly via methylation. Modification patterns seen in the chromatin sample are not seen in unmethylated oligonucleosomal DNA or fully methylated K562 oligonucleosomal DNA.

    Journal: bioRxiv

    Article Title: Massively multiplex single-molecule oligonucleosome footprinting

    doi: 10.1101/2020.05.20.105379

    Figure Lengend Snippet: In vivo SAMOSA captures oligonucleosome structure by combining MNase digestion of chromatin with adenine methylation footprinting. A.) An overview of the in vivo SAMOSA protocol: oligonucleosomes are gently solubilized from nuclei using micrococcal nuclease and fusogenic lipid treatment. Resulting oligonucleosomes are footprinted using the EcoGII enzyme and sequencing on the PacBio platform. Each sequencing molecules captures two orthogonal biological signals: MNase cuts that capture ‘barrier’ protein-DNA interactions, and m 6 dA methylation protein-DNA footprints. B.) Fragment length distributions for in vivo SAMOSA data reveal expected oligonucleosomal laddering (bin size = 5 bp). C.) Averaged modification probabilities from SAMOSA experiments demonstrate the ability to mark nucleosome-DNA interactions directly via methylation. Modification patterns seen in the chromatin sample are not seen in unmethylated oligonucleosomal DNA or fully methylated K562 oligonucleosomal DNA.

    Article Snippet: Isolation of nuclei, MNase digest, and overnight dialysis 100E6 K562 cells were collected by centrifugation (300x g , 5 min), washed in ice cold 1X PBS, and resuspended in 1 mL Nuclear Isolation Buffer (20mM HEPES, 10mM KCl, 1mM MgCl2, 0.1% Triton X-100, 20% Glycerol, and 1X Protease Inhibitor (Roche)) per 5-10 e6 cells by gently pipetting 5x with a wide-bore tip to release nuclei.

    Techniques: In Vivo, Methylation, Footprinting, Sequencing, Modification

    Bulk chromatin and centromeric chromatin were solubilized by MNase digestion of HeLa nuclei in 0.3 M NaCl. (A) Centromeric proteins CENP-A, -B, and -C were solubilized by MNase digestion. Isolated HeLa nuclei (2 × 10 8 ) were suspended with 1 ml of WB containing 0.3 M NaCl (sample a in lane 1 and sample c in lanes 4 to 6) or 0.6 M NaCl (sample b in lanes 2 and 3). Sample c was digested with 60 U of MNase per ml for 10 min at 37°C. Soluble and insoluble materials from each sample were separated by centrifugation. ACA beads were added to the supernatant of sample c and incubated overnight at 4°C. Pellets were resuspended in 1 ml of SDS buffer by extensive sonication and 5 μl of each sample was separated by SDS-7.5% (for CENP-B and CENP-C) or 12.5% (for CENP-A) PAGE, and centromeric proteins were detected by immunostaining with ACA serum. Lane 1, supernatant fraction of a; lane 2, supernatant fraction of b; lane 3, pellet fraction of b; lane 4, supernatant fraction of c before addition of ACA beads; lane 5, supernatant fraction of c after addition of ACA beads; lane 6, pellet fraction of c. Lane M, marker centromeric proteins, CENP-A, CENP-B, and CENP-C. (B) Size distribution of DNA fragments from bulk chromatin after MNase digestion. HeLa nuclei were digested with MNase to various extents. The fragmented DNA in the soluble fractions was extracted with phenol and electrophoresed through 1% agarose gel. DNA was detected with ethidium bromide staining. Lane 1, 20 U/ml for 2 min (40 U/ml × min, sample 1); lane 2, 20 U/ml for 4 min (80 U/ml × min, sample 2); lane 3, 40 U/ml for 5 min (200 U/ml × min, sample 3); lane 4, 80 U/ml for 45 min (3,600 U/ml × min, sample 4). Positions of the DNA size markers are indicated at the left. (C) Detection of core histones and CENP-A in each fraction. Soluble (sup.) and insoluble (pellet) fractions were subjected to SDS-12.5% PAGE, and the separated core histones were stained with Coomassie brilliant blue (upper panel). The proteins were transferred to a membrane and immunolabeled with ACA serum (AK) (lower panel). Lane M in the lower panel is a recombinant CENP-A marker protein. Lanes 1 to 4 correspond to samples 1 to 4 of the soluble (sup.) fractions, and lanes 5 to 8 to samples 1 to 4 of the pellet fractions.

    Journal: Molecular and Cellular Biology

    Article Title: CENP-A, -B, and -C Chromatin Complex That Contains the I-Type ?-Satellite Array Constitutes the Prekinetochore in HeLa Cells

    doi: 10.1128/MCB.22.7.2229-2241.2002

    Figure Lengend Snippet: Bulk chromatin and centromeric chromatin were solubilized by MNase digestion of HeLa nuclei in 0.3 M NaCl. (A) Centromeric proteins CENP-A, -B, and -C were solubilized by MNase digestion. Isolated HeLa nuclei (2 × 10 8 ) were suspended with 1 ml of WB containing 0.3 M NaCl (sample a in lane 1 and sample c in lanes 4 to 6) or 0.6 M NaCl (sample b in lanes 2 and 3). Sample c was digested with 60 U of MNase per ml for 10 min at 37°C. Soluble and insoluble materials from each sample were separated by centrifugation. ACA beads were added to the supernatant of sample c and incubated overnight at 4°C. Pellets were resuspended in 1 ml of SDS buffer by extensive sonication and 5 μl of each sample was separated by SDS-7.5% (for CENP-B and CENP-C) or 12.5% (for CENP-A) PAGE, and centromeric proteins were detected by immunostaining with ACA serum. Lane 1, supernatant fraction of a; lane 2, supernatant fraction of b; lane 3, pellet fraction of b; lane 4, supernatant fraction of c before addition of ACA beads; lane 5, supernatant fraction of c after addition of ACA beads; lane 6, pellet fraction of c. Lane M, marker centromeric proteins, CENP-A, CENP-B, and CENP-C. (B) Size distribution of DNA fragments from bulk chromatin after MNase digestion. HeLa nuclei were digested with MNase to various extents. The fragmented DNA in the soluble fractions was extracted with phenol and electrophoresed through 1% agarose gel. DNA was detected with ethidium bromide staining. Lane 1, 20 U/ml for 2 min (40 U/ml × min, sample 1); lane 2, 20 U/ml for 4 min (80 U/ml × min, sample 2); lane 3, 40 U/ml for 5 min (200 U/ml × min, sample 3); lane 4, 80 U/ml for 45 min (3,600 U/ml × min, sample 4). Positions of the DNA size markers are indicated at the left. (C) Detection of core histones and CENP-A in each fraction. Soluble (sup.) and insoluble (pellet) fractions were subjected to SDS-12.5% PAGE, and the separated core histones were stained with Coomassie brilliant blue (upper panel). The proteins were transferred to a membrane and immunolabeled with ACA serum (AK) (lower panel). Lane M in the lower panel is a recombinant CENP-A marker protein. Lanes 1 to 4 correspond to samples 1 to 4 of the soluble (sup.) fractions, and lanes 5 to 8 to samples 1 to 4 of the pellet fractions.

    Article Snippet: The nuclear suspension was digested with MNase (Roche Diagnostics) at 37°C after addition of CaCl2 to a final concentration of 2 mM.

    Techniques: Isolation, Western Blot, Centrifugation, Incubation, Sonication, Polyacrylamide Gel Electrophoresis, Immunostaining, Marker, Agarose Gel Electrophoresis, Staining, Immunolabeling, Recombinant

    Nuclease and PK digestion of fractionated and unfractionated NCs. (A) Sucrose gradient fractions 6, 9, and 12 prepared from lysates without prior MNase treatment () were digested with MNase or DNase I as described in Materials and Methods. The reaction

    Journal: Journal of Virology

    Article Title: Maturation-Associated Destabilization of Hepatitis B Virus Nucleocapsid

    doi: 10.1128/JVI.01912-13

    Figure Lengend Snippet: Nuclease and PK digestion of fractionated and unfractionated NCs. (A) Sucrose gradient fractions 6, 9, and 12 prepared from lysates without prior MNase treatment () were digested with MNase or DNase I as described in Materials and Methods. The reaction

    Article Snippet: NCs isolated by sucrose gradient centrifugation (10 μl of the indicated sucrose fraction) or cytoplasmic lysate (10 μl) were treated with 1 mg/ml of PK (Invitrogen) (unless noted otherwise) in EPR buffer in a total volume of 20 μl at 37°C for 1 h. Similarly, for nuclease treatment, MNase (0.25 unit/μl) or DNase I (Roche) (2 mg/ml) was added, and the reaction mixture was incubated at 37°C for 1 h. For MNase digestion, the MgCl2 in the EPR buffer was replaced with CaCl2 (5 mM).

    Techniques:

    Humanized yeast have trouble adapting to new conditions (A) Violin plots showing that humanized yeast cells are larger and have dysregulated cell size based on phase-contrast microscopy measurements. (B) Cell-cycle ( CLB1 ) and cell-size ( WHI4 ) regulating genes each have highly occupied −1 and −2 nucleosomes. (C) Humanized yeast have a prolonged S-phase and/or arrest in G1. Cell-cycle was analyzed by sytox green straining of DNA content and measured by flow cytometry. Each plot shows 10,000 cells. (D) Humanized yeast have delayed remodeling at the GAL1 promoter. Time-course was analyzed by galactose induction of eGFP using flow cytometry. (E) MNase-seq map of PHO5 promoter, and time-course nucleosome scanning assay using WT, hH3.1-core, and hH3.3-core nucleosome yeasts upon phosphate starvation at different time points. Data points show qPCR amplicon midpoints, and mean ± SD of 2 biological replicates.

    Journal: Cell

    Article Title: Resetting the yeast epigenome with human nucleosomes

    doi: 10.1016/j.cell.2017.10.043

    Figure Lengend Snippet: Humanized yeast have trouble adapting to new conditions (A) Violin plots showing that humanized yeast cells are larger and have dysregulated cell size based on phase-contrast microscopy measurements. (B) Cell-cycle ( CLB1 ) and cell-size ( WHI4 ) regulating genes each have highly occupied −1 and −2 nucleosomes. (C) Humanized yeast have a prolonged S-phase and/or arrest in G1. Cell-cycle was analyzed by sytox green straining of DNA content and measured by flow cytometry. Each plot shows 10,000 cells. (D) Humanized yeast have delayed remodeling at the GAL1 promoter. Time-course was analyzed by galactose induction of eGFP using flow cytometry. (E) MNase-seq map of PHO5 promoter, and time-course nucleosome scanning assay using WT, hH3.1-core, and hH3.3-core nucleosome yeasts upon phosphate starvation at different time points. Data points show qPCR amplicon midpoints, and mean ± SD of 2 biological replicates.

    Article Snippet: Processed MNase digested DNA was analyzed by qPCR using primers listed in in a Roche LightCycler 1536 real-time PCR machine using LightCycler 1536 DNA Green Master (Roche) in technical triplicate.

    Techniques: Microscopy, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Amplification