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Bio-Rad dnase i
Dnase I, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 98/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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dnase i - by Bioz Stars, 2020-04
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Real-time Polymerase Chain Reaction:

Article Title: Dual activity of niclosamide to suppress replication of integrated HIV-1 and Mycobacterium tuberculosis (Beijing).
Article Snippet: .. qPCR RNA was isolated using a Quick-RNA kit (Zymo), treated with DNase I, and cDNA synthesized with an iScript cDNA kit (BioRad). .. Semi-quantitative RT-PCR was performed to detect HIV LTR as previously described [29] using iTaq SYBR Green master mix (BioRad) on a LightCycler instrument (BioRad) and normalized to the GAPDH housekeeping gene.

Article Title: Elucidation of the Regulon and cis-Acting Regulatory Element of HrpB, the AraC-Type Regulator of a Plant Pathogen-Like Type III Secretion System in Burkholderia pseudomallei ▿ ▿ †
Article Snippet: After an equal volume of RNAprotect (Qiagen) was added to the culture, RNA was purified from pelleted cells by using the RNeasy kit (Qiagen) and treated with DNase I (Qiagen) according to manufacturer's instructions, except that the amount of DNase I was increased 3-fold. cDNA was synthesized using 1 μg of RNA and the iScript cDNA synthesis kit (Bio-Rad) following the manufacturer's instructions. .. Quantitative PCR was performed on 22 target genes with an Applied Biosystems StepOne real-time PCR system using Sybr green under the following conditions: 10 min at 95°C and 40 cycles of 95°C for 15 s and 60°C for 1 min.

Amplification:

Article Title: Elucidation of the Regulon and cis-Acting Regulatory Element of HrpB, the AraC-Type Regulator of a Plant Pathogen-Like Type III Secretion System in Burkholderia pseudomallei ▿ ▿ †
Article Snippet: After an equal volume of RNAprotect (Qiagen) was added to the culture, RNA was purified from pelleted cells by using the RNeasy kit (Qiagen) and treated with DNase I (Qiagen) according to manufacturer's instructions, except that the amount of DNase I was increased 3-fold. cDNA was synthesized using 1 μg of RNA and the iScript cDNA synthesis kit (Bio-Rad) following the manufacturer's instructions. .. The amplification data were analyzed by the 2−ΔΔCT method to determine relative log2 changes in gene expression ( ) in reference to rpoA for B. pseudomallei and B. thailandensis and the cytochrome oxidase gene (RSc0369) for R. solanacearum .

Synthesized:

Article Title: Dual activity of niclosamide to suppress replication of integrated HIV-1 and Mycobacterium tuberculosis (Beijing).
Article Snippet: .. qPCR RNA was isolated using a Quick-RNA kit (Zymo), treated with DNase I, and cDNA synthesized with an iScript cDNA kit (BioRad). .. Semi-quantitative RT-PCR was performed to detect HIV LTR as previously described [29] using iTaq SYBR Green master mix (BioRad) on a LightCycler instrument (BioRad) and normalized to the GAPDH housekeeping gene.

Article Title: Elucidation of the Regulon and cis-Acting Regulatory Element of HrpB, the AraC-Type Regulator of a Plant Pathogen-Like Type III Secretion System in Burkholderia pseudomallei ▿ ▿ †
Article Snippet: .. After an equal volume of RNAprotect (Qiagen) was added to the culture, RNA was purified from pelleted cells by using the RNeasy kit (Qiagen) and treated with DNase I (Qiagen) according to manufacturer's instructions, except that the amount of DNase I was increased 3-fold. cDNA was synthesized using 1 μg of RNA and the iScript cDNA synthesis kit (Bio-Rad) following the manufacturer's instructions. .. Quantitative PCR was performed on 22 target genes with an Applied Biosystems StepOne real-time PCR system using Sybr green under the following conditions: 10 min at 95°C and 40 cycles of 95°C for 15 s and 60°C for 1 min.

Isolation:

Article Title: Dual activity of niclosamide to suppress replication of integrated HIV-1 and Mycobacterium tuberculosis (Beijing).
Article Snippet: .. qPCR RNA was isolated using a Quick-RNA kit (Zymo), treated with DNase I, and cDNA synthesized with an iScript cDNA kit (BioRad). .. Semi-quantitative RT-PCR was performed to detect HIV LTR as previously described [29] using iTaq SYBR Green master mix (BioRad) on a LightCycler instrument (BioRad) and normalized to the GAPDH housekeeping gene.

Quantitative RT-PCR:

Article Title: Elucidation of the Regulon and cis-Acting Regulatory Element of HrpB, the AraC-Type Regulator of a Plant Pathogen-Like Type III Secretion System in Burkholderia pseudomallei ▿ ▿ †
Article Snippet: Paragraph title: Quantitative reverse transcription PCR (qRT-PCR). ... After an equal volume of RNAprotect (Qiagen) was added to the culture, RNA was purified from pelleted cells by using the RNeasy kit (Qiagen) and treated with DNase I (Qiagen) according to manufacturer's instructions, except that the amount of DNase I was increased 3-fold. cDNA was synthesized using 1 μg of RNA and the iScript cDNA synthesis kit (Bio-Rad) following the manufacturer's instructions.

Purification:

Article Title: Elucidation of the Regulon and cis-Acting Regulatory Element of HrpB, the AraC-Type Regulator of a Plant Pathogen-Like Type III Secretion System in Burkholderia pseudomallei ▿ ▿ †
Article Snippet: .. After an equal volume of RNAprotect (Qiagen) was added to the culture, RNA was purified from pelleted cells by using the RNeasy kit (Qiagen) and treated with DNase I (Qiagen) according to manufacturer's instructions, except that the amount of DNase I was increased 3-fold. cDNA was synthesized using 1 μg of RNA and the iScript cDNA synthesis kit (Bio-Rad) following the manufacturer's instructions. .. Quantitative PCR was performed on 22 target genes with an Applied Biosystems StepOne real-time PCR system using Sybr green under the following conditions: 10 min at 95°C and 40 cycles of 95°C for 15 s and 60°C for 1 min.

SYBR Green Assay:

Article Title: Dual activity of niclosamide to suppress replication of integrated HIV-1 and Mycobacterium tuberculosis (Beijing).
Article Snippet: qPCR RNA was isolated using a Quick-RNA kit (Zymo), treated with DNase I, and cDNA synthesized with an iScript cDNA kit (BioRad). .. Semi-quantitative RT-PCR was performed to detect HIV LTR as previously described [29] using iTaq SYBR Green master mix (BioRad) on a LightCycler instrument (BioRad) and normalized to the GAPDH housekeeping gene.

Article Title: Elucidation of the Regulon and cis-Acting Regulatory Element of HrpB, the AraC-Type Regulator of a Plant Pathogen-Like Type III Secretion System in Burkholderia pseudomallei ▿ ▿ †
Article Snippet: After an equal volume of RNAprotect (Qiagen) was added to the culture, RNA was purified from pelleted cells by using the RNeasy kit (Qiagen) and treated with DNase I (Qiagen) according to manufacturer's instructions, except that the amount of DNase I was increased 3-fold. cDNA was synthesized using 1 μg of RNA and the iScript cDNA synthesis kit (Bio-Rad) following the manufacturer's instructions. .. Quantitative PCR was performed on 22 target genes with an Applied Biosystems StepOne real-time PCR system using Sybr green under the following conditions: 10 min at 95°C and 40 cycles of 95°C for 15 s and 60°C for 1 min.

Reverse Transcription Polymerase Chain Reaction:

Article Title: Dual activity of niclosamide to suppress replication of integrated HIV-1 and Mycobacterium tuberculosis (Beijing).
Article Snippet: qPCR RNA was isolated using a Quick-RNA kit (Zymo), treated with DNase I, and cDNA synthesized with an iScript cDNA kit (BioRad). .. Semi-quantitative RT-PCR was performed to detect HIV LTR as previously described [29] using iTaq SYBR Green master mix (BioRad) on a LightCycler instrument (BioRad) and normalized to the GAPDH housekeeping gene.

Incubation:

Article Title: Elucidation of the Regulon and cis-Acting Regulatory Element of HrpB, the AraC-Type Regulator of a Plant Pathogen-Like Type III Secretion System in Burkholderia pseudomallei ▿ ▿ †
Article Snippet: Rhamnose was added to 0.2% to induce HrpBbp expression, and incubation continued until the OD600 was 1.2. .. After an equal volume of RNAprotect (Qiagen) was added to the culture, RNA was purified from pelleted cells by using the RNeasy kit (Qiagen) and treated with DNase I (Qiagen) according to manufacturer's instructions, except that the amount of DNase I was increased 3-fold. cDNA was synthesized using 1 μg of RNA and the iScript cDNA synthesis kit (Bio-Rad) following the manufacturer's instructions.

Expressing:

Article Title: Elucidation of the Regulon and cis-Acting Regulatory Element of HrpB, the AraC-Type Regulator of a Plant Pathogen-Like Type III Secretion System in Burkholderia pseudomallei ▿ ▿ †
Article Snippet: Rhamnose was added to 0.2% to induce HrpBbp expression, and incubation continued until the OD600 was 1.2. .. After an equal volume of RNAprotect (Qiagen) was added to the culture, RNA was purified from pelleted cells by using the RNeasy kit (Qiagen) and treated with DNase I (Qiagen) according to manufacturer's instructions, except that the amount of DNase I was increased 3-fold. cDNA was synthesized using 1 μg of RNA and the iScript cDNA synthesis kit (Bio-Rad) following the manufacturer's instructions.

Polymerase Chain Reaction:

Article Title: Elucidation of the Regulon and cis-Acting Regulatory Element of HrpB, the AraC-Type Regulator of a Plant Pathogen-Like Type III Secretion System in Burkholderia pseudomallei ▿ ▿ †
Article Snippet: Paragraph title: Quantitative reverse transcription PCR (qRT-PCR). ... After an equal volume of RNAprotect (Qiagen) was added to the culture, RNA was purified from pelleted cells by using the RNeasy kit (Qiagen) and treated with DNase I (Qiagen) according to manufacturer's instructions, except that the amount of DNase I was increased 3-fold. cDNA was synthesized using 1 μg of RNA and the iScript cDNA synthesis kit (Bio-Rad) following the manufacturer's instructions.

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  • 88
    Bio-Rad dnase i sensitivity assay
    Noisier clones exhibit more inaccessible promoters A HIV-1 LTR exhibits positioned nucleosomes. Schematic figure showing the well-characterized placement of nucleosomes along the HIV-1 promoter. Nuc-1 is positioned at the transcription start site (TSS), and Nuc-0 is placed further upstream along the promoter. B Significant differences in DNA accessibility around TSS between high and low noise clones. The region highlighted in black was probed after digestion with <t>DNase</t> I for 16 clones. The figure plots the level of chromatin inaccessibility for pairs of clones that exhibit similar mean levels of expression. Clones that exhibit noisier gene expression also have more closed chromatin. C–E Differential DNA accessibility between high and low noise clones across three distinct sites in the LTR. The HIV-1 promoter was probed in greater detail with the color scheme matching that shown in (A). As in (B), it was found that for similar mean levels of gene expression, noisier clones exhibited more closed chromatin along the entire length of the promoter (ratios > 1 in all cases). Interestingly, compared to the two other regions of the promoter, the hypersensitive site (HSS) showed maximum differences in chromatin inaccessibility between high and low noise clones. Data information: qPCR experiments were performed in triplicate, and the error bars reflect the standard deviation from the mean.
    Dnase I Sensitivity Assay, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnase i sensitivity assay/product/Bio-Rad
    Average 88 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    dnase i sensitivity assay - by Bioz Stars, 2020-04
    88/100 stars
      Buy from Supplier

    99
    Bio-Rad dnase i
    Confirmatory real-time PCR analysis ( a–f ) DNase-chip–identified regions were confirmed by real-time PCR for CD4 + T cells ( a,c,e ) and GM06990 cells ( b,d,f (MPSS cluster). Real-time PCR using primer sets flanking DNase-chip peaks that are present with all three <t>DNase</t> I concentrations ( a,b ). Real-time PCR using primers sets flanking DNase-chip peaks that are present with two out of three DNase I concentrations ( c,d ). Real-time PCR using primer sets flanking DNase-chip peaks that are present with only a single DNase I concentration ( e,f ).
    Dnase I, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnase i/product/Bio-Rad
    Average 99 stars, based on 53 article reviews
    Price from $9.99 to $1999.99
    dnase i - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    Image Search Results


    Noisier clones exhibit more inaccessible promoters A HIV-1 LTR exhibits positioned nucleosomes. Schematic figure showing the well-characterized placement of nucleosomes along the HIV-1 promoter. Nuc-1 is positioned at the transcription start site (TSS), and Nuc-0 is placed further upstream along the promoter. B Significant differences in DNA accessibility around TSS between high and low noise clones. The region highlighted in black was probed after digestion with DNase I for 16 clones. The figure plots the level of chromatin inaccessibility for pairs of clones that exhibit similar mean levels of expression. Clones that exhibit noisier gene expression also have more closed chromatin. C–E Differential DNA accessibility between high and low noise clones across three distinct sites in the LTR. The HIV-1 promoter was probed in greater detail with the color scheme matching that shown in (A). As in (B), it was found that for similar mean levels of gene expression, noisier clones exhibited more closed chromatin along the entire length of the promoter (ratios > 1 in all cases). Interestingly, compared to the two other regions of the promoter, the hypersensitive site (HSS) showed maximum differences in chromatin inaccessibility between high and low noise clones. Data information: qPCR experiments were performed in triplicate, and the error bars reflect the standard deviation from the mean.

    Journal: Molecular Systems Biology

    Article Title: Orthogonal control of expression mean and variance by epigenetic features at different genomic loci

    doi: 10.15252/msb.20145704

    Figure Lengend Snippet: Noisier clones exhibit more inaccessible promoters A HIV-1 LTR exhibits positioned nucleosomes. Schematic figure showing the well-characterized placement of nucleosomes along the HIV-1 promoter. Nuc-1 is positioned at the transcription start site (TSS), and Nuc-0 is placed further upstream along the promoter. B Significant differences in DNA accessibility around TSS between high and low noise clones. The region highlighted in black was probed after digestion with DNase I for 16 clones. The figure plots the level of chromatin inaccessibility for pairs of clones that exhibit similar mean levels of expression. Clones that exhibit noisier gene expression also have more closed chromatin. C–E Differential DNA accessibility between high and low noise clones across three distinct sites in the LTR. The HIV-1 promoter was probed in greater detail with the color scheme matching that shown in (A). As in (B), it was found that for similar mean levels of gene expression, noisier clones exhibited more closed chromatin along the entire length of the promoter (ratios > 1 in all cases). Interestingly, compared to the two other regions of the promoter, the hypersensitive site (HSS) showed maximum differences in chromatin inaccessibility between high and low noise clones. Data information: qPCR experiments were performed in triplicate, and the error bars reflect the standard deviation from the mean.

    Article Snippet: DNase I sensitivity assay The assay was performed using the EpiQ Chromatin Analysis Kit (Bio-Rad) as previously described (Dey et al , ; Miller-Jensen et al , ).

    Techniques: Clone Assay, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

    Confirmatory real-time PCR analysis ( a–f ) DNase-chip–identified regions were confirmed by real-time PCR for CD4 + T cells ( a,c,e ) and GM06990 cells ( b,d,f (MPSS cluster). Real-time PCR using primer sets flanking DNase-chip peaks that are present with all three DNase I concentrations ( a,b ). Real-time PCR using primers sets flanking DNase-chip peaks that are present with two out of three DNase I concentrations ( c,d ). Real-time PCR using primer sets flanking DNase-chip peaks that are present with only a single DNase I concentration ( e,f ).

    Journal: Nature methods

    Article Title: DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays

    doi: 10.1038/NMETH888

    Figure Lengend Snippet: Confirmatory real-time PCR analysis ( a–f ) DNase-chip–identified regions were confirmed by real-time PCR for CD4 + T cells ( a,c,e ) and GM06990 cells ( b,d,f (MPSS cluster). Real-time PCR using primer sets flanking DNase-chip peaks that are present with all three DNase I concentrations ( a,b ). Real-time PCR using primers sets flanking DNase-chip peaks that are present with two out of three DNase I concentrations ( c,d ). Real-time PCR using primer sets flanking DNase-chip peaks that are present with only a single DNase I concentration ( e,f ).

    Article Snippet: We used pulsed field gel electrophoresis (pulse time, 20–60 s for 18 h) to identify the three concentrations of DNase I to be used for DNase-chip (Bio-Rad).

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

    Location of DNase I hypersensitive sites relative to the annotated genome ( a,b ) DNase-chip peaks were mapped to ENCODE regions stratified by gene density and human-mouse sequence conservation for both CD4 + T cells ( a ) and the GM06990 lymphoblastoid cell line ( b ). ( c ) The genomic locations of DNase I hypersensitive sites (detected with at least two concentrations of DNase I) and computationally generated random controls ( n = 1,000) were compared to Gencode transcription start and end sites (within a 2-kb window), CpG islands, first introns, non-first introns, first exons, non-first exons, conserved sequences (MCS), conserved sequences minus coding exons (MCS-no-CDS). The number of DNase I hypersensitive sites at different distances (0 kb, 2 kb, 10 kb, and 25 kb) from Gencode genes was also determined. Error bars represent the entire range of values seen randomly generated mock datasets ( n = 1,000). Compared to the random controls, the locations of the DNase-chip peaks are significantly (Monte Carlo P

    Journal: Nature methods

    Article Title: DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays

    doi: 10.1038/NMETH888

    Figure Lengend Snippet: Location of DNase I hypersensitive sites relative to the annotated genome ( a,b ) DNase-chip peaks were mapped to ENCODE regions stratified by gene density and human-mouse sequence conservation for both CD4 + T cells ( a ) and the GM06990 lymphoblastoid cell line ( b ). ( c ) The genomic locations of DNase I hypersensitive sites (detected with at least two concentrations of DNase I) and computationally generated random controls ( n = 1,000) were compared to Gencode transcription start and end sites (within a 2-kb window), CpG islands, first introns, non-first introns, first exons, non-first exons, conserved sequences (MCS), conserved sequences minus coding exons (MCS-no-CDS). The number of DNase I hypersensitive sites at different distances (0 kb, 2 kb, 10 kb, and 25 kb) from Gencode genes was also determined. Error bars represent the entire range of values seen randomly generated mock datasets ( n = 1,000). Compared to the random controls, the locations of the DNase-chip peaks are significantly (Monte Carlo P

    Article Snippet: We used pulsed field gel electrophoresis (pulse time, 20–60 s for 18 h) to identify the three concentrations of DNase I to be used for DNase-chip (Bio-Rad).

    Techniques: Chromatin Immunoprecipitation, Sequencing, Generated

    Identification of cell type–specific DNase I hypersensitive sites ( a,b ) Real-time PCR was performed on both CD4 + T cells and GM06990 cells. Real-time PCR using primer sets that flank random regions of the genome or DNase-chip peaks that are present for both cell types ( a ). Real-time PCR using primer sets that flank DNase-chip peaks that are present in only CD4 + T cells (CD4) or GM06990 cells (GM; b ).

    Journal: Nature methods

    Article Title: DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays

    doi: 10.1038/NMETH888

    Figure Lengend Snippet: Identification of cell type–specific DNase I hypersensitive sites ( a,b ) Real-time PCR was performed on both CD4 + T cells and GM06990 cells. Real-time PCR using primer sets that flank random regions of the genome or DNase-chip peaks that are present for both cell types ( a ). Real-time PCR using primer sets that flank DNase-chip peaks that are present in only CD4 + T cells (CD4) or GM06990 cells (GM; b ).

    Article Snippet: We used pulsed field gel electrophoresis (pulse time, 20–60 s for 18 h) to identify the three concentrations of DNase I to be used for DNase-chip (Bio-Rad).

    Techniques: Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

    Expression of genes relative to proximity to DNase I hypersensitive sites ( a,b ) The distance of each transcription start site (blue dots) to the nearest DNase I hypersensitive site was compared to the gene expression values of each transcript for both CD4 + T cells ( a ) and the GM06990 lymphoblastoid cell line ( b ). Horizontal red lines mark the expression level that separates most genes that have a DNase I hypersensitive site nearby (

    Journal: Nature methods

    Article Title: DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays

    doi: 10.1038/NMETH888

    Figure Lengend Snippet: Expression of genes relative to proximity to DNase I hypersensitive sites ( a,b ) The distance of each transcription start site (blue dots) to the nearest DNase I hypersensitive site was compared to the gene expression values of each transcript for both CD4 + T cells ( a ) and the GM06990 lymphoblastoid cell line ( b ). Horizontal red lines mark the expression level that separates most genes that have a DNase I hypersensitive site nearby (

    Article Snippet: We used pulsed field gel electrophoresis (pulse time, 20–60 s for 18 h) to identify the three concentrations of DNase I to be used for DNase-chip (Bio-Rad).

    Techniques: Expressing

    DNase-chip protocol ( a ) Pulsed field gel electrophoresis of DNase I–digested nuclear DNA. The concentrations of DNase I used for DNase-chip are labeled as A, B and C. ( b ) Outline of DNase-chip protocol. ( c ) Histogram of signal ratios of DNase I–treated versus random-sheared DNA. Tiled oligos that displayed the top 5% ratios are located to the right of the red bar. ( d ) Identification of regions with significant P values. The raw ratio data are plotted in gray, with the y-axis label on the right; the top 5% cutoff is displayed as a dotted horizontal gray line. The P value data for sliding 500-bp windows are plotted in red, with the y -axis label on the left.

    Journal: Nature methods

    Article Title: DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays

    doi: 10.1038/NMETH888

    Figure Lengend Snippet: DNase-chip protocol ( a ) Pulsed field gel electrophoresis of DNase I–digested nuclear DNA. The concentrations of DNase I used for DNase-chip are labeled as A, B and C. ( b ) Outline of DNase-chip protocol. ( c ) Histogram of signal ratios of DNase I–treated versus random-sheared DNA. Tiled oligos that displayed the top 5% ratios are located to the right of the red bar. ( d ) Identification of regions with significant P values. The raw ratio data are plotted in gray, with the y-axis label on the right; the top 5% cutoff is displayed as a dotted horizontal gray line. The P value data for sliding 500-bp windows are plotted in red, with the y -axis label on the left.

    Article Snippet: We used pulsed field gel electrophoresis (pulse time, 20–60 s for 18 h) to identify the three concentrations of DNase I to be used for DNase-chip (Bio-Rad).

    Techniques: Chromatin Immunoprecipitation, Pulsed-Field Gel, Electrophoresis, Labeling

    TALEs facilitate displacement of a positioned nucleosome on the IL-2 promoter. (A) Proposed TALE mechanism upon binding to the IL-2 promoter (not drawn to scale). Top panel is an illustration of the proposed chromatin structure of the IL-2 promoter in the absence of TALE activators. The approximate location of response elements adapted from previously published data [31] . Circle represents a positioned nucleosome located approximately 60 to 200 bp upstream of the TSS [31] . Boxed enclosed ‘T’ represents the TATA box. Bottom panel indicates location of TALE activators relative to TSS and proposed mechanism of action on the IL-2 promoter. IL-2 specific primers were used to probe various regions across the IL-2 promoter. Arrows indicate the approximate location of each region amplified by their corresponding primer set. All TALE activators were fused to VP64 (activation domain colored green) with the exception of IL2D’, which was fused to TBP (activation domain colored red). (B) Results of chromatin analysis using CHART-PCR. Percent accessibility was calculated as described in Methods and plotted for both empty vector control (black bars) and TALE activators (shaded bars). GAPDH used as internal control to monitor DNase I digestion. Results shown are of three independent experiments (error bars, mean +/− SD, n = 3). Statistical analysis was determined using one tailed, Welch’s t -test (P

    Journal: PLoS ONE

    Article Title: Activation of Silenced Cytokine Gene Promoters by the Synergistic Effect of TBP-TALE and VP64-TALE Activators

    doi: 10.1371/journal.pone.0095790

    Figure Lengend Snippet: TALEs facilitate displacement of a positioned nucleosome on the IL-2 promoter. (A) Proposed TALE mechanism upon binding to the IL-2 promoter (not drawn to scale). Top panel is an illustration of the proposed chromatin structure of the IL-2 promoter in the absence of TALE activators. The approximate location of response elements adapted from previously published data [31] . Circle represents a positioned nucleosome located approximately 60 to 200 bp upstream of the TSS [31] . Boxed enclosed ‘T’ represents the TATA box. Bottom panel indicates location of TALE activators relative to TSS and proposed mechanism of action on the IL-2 promoter. IL-2 specific primers were used to probe various regions across the IL-2 promoter. Arrows indicate the approximate location of each region amplified by their corresponding primer set. All TALE activators were fused to VP64 (activation domain colored green) with the exception of IL2D’, which was fused to TBP (activation domain colored red). (B) Results of chromatin analysis using CHART-PCR. Percent accessibility was calculated as described in Methods and plotted for both empty vector control (black bars) and TALE activators (shaded bars). GAPDH used as internal control to monitor DNase I digestion. Results shown are of three independent experiments (error bars, mean +/− SD, n = 3). Statistical analysis was determined using one tailed, Welch’s t -test (P

    Article Snippet: To determine percent accessibility to DNase I digestion, the EpiQ chromatin analysis tool (Bio-Rad) was used to analyze each sample using the following equation: % Accessibility to DNase I = (1 − 2ΔCt ) × 100.

    Techniques: Binding Assay, Amplification, Activation Assay, Polymerase Chain Reaction, Plasmid Preparation, One-tailed Test

    Overview of the approach. ( A ) Strategy. HUVECs were stimulated ± TNFα for 0 or 30 min, and harvested; intact nuclei were isolated in a physiological buffer and digested with DNase I. After spinning, most chromatin (and ‘chromatin-associated’ RNA) is released into the supernatant. The resulting pellet was treated with a mixture of caspases to detach factories (red) from the underlying sub-structure (brown line). Following centrifugation, the supernatant yields ‘factory’ RNA, whilst the pellet contains factory remnants and residual chromatin. Total (ribodepleted), and poly(A) + -enriched RNA fractions were also collected. ( B ) Genome browser view of a typical, constitutively-expressed, gene ( EDN1 ) illustrating read densities obtained by sequencing total, factory and poly(A) +  RNA. ( C ) Average coverage profiles from two biological replicates along 2722 and 2386 concatenated exons and introns, respectively, belonging to 309 TNFα-responsive genes. Plots also include 100 bp up/downstream of exons/introns.

    Journal: Nucleic Acids Research

    Article Title: Dissecting the nascent human transcriptome by analysing the RNA content of transcription factories

    doi: 10.1093/nar/gkv390

    Figure Lengend Snippet: Overview of the approach. ( A ) Strategy. HUVECs were stimulated ± TNFα for 0 or 30 min, and harvested; intact nuclei were isolated in a physiological buffer and digested with DNase I. After spinning, most chromatin (and ‘chromatin-associated’ RNA) is released into the supernatant. The resulting pellet was treated with a mixture of caspases to detach factories (red) from the underlying sub-structure (brown line). Following centrifugation, the supernatant yields ‘factory’ RNA, whilst the pellet contains factory remnants and residual chromatin. Total (ribodepleted), and poly(A) + -enriched RNA fractions were also collected. ( B ) Genome browser view of a typical, constitutively-expressed, gene ( EDN1 ) illustrating read densities obtained by sequencing total, factory and poly(A) + RNA. ( C ) Average coverage profiles from two biological replicates along 2722 and 2386 concatenated exons and introns, respectively, belonging to 309 TNFα-responsive genes. Plots also include 100 bp up/downstream of exons/introns.

    Article Snippet: Then, following treatment with DNase I and caspases as above, released ‘factory’ complexes were resolved in two-dimensional acrylamide-agarose composite gels using the Mini-Protean 3 system (BioRad); distributions of nascent transcripts was visualized by autoradiography, while those of RNA polymerase II by western blotting using the iBlot transfer system (Invitrogen) and a mouse monoclonal antibody against its RPB1 subunit (7C2; ref. )—all as described in ref. ( ).

    Techniques: Isolation, Centrifugation, Sequencing