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  • 99
    New England Biolabs lambda exonuclease digestion
    Superior performance of ChIP-nexus in discovering relevant binding footprints for transcription factors (a) Outline of ChIP-nexus 1) The transcription factor of interest (brown) is immunoprecipitated from chromatin fragments with antibodies in the same way as during conventional ChIP-seq experiments. 2) While still bound to the antibodies, the DNA ends are repaired, dA-tailed and then ligated to a special adaptor that contains a pair of sequences for library amplification (arrows indicate the correct orientation for them to be functional), a BamHI site (black dot) for linearization, and a 9-nucleotide barcode containing 5 random bases and 4 fixed bases to remove reads resulting from over-amplification of library DNA. The barcode is part of a 5′ overhang, which reduces adaptor-adaptor ligation. 3) After the adaptor ligation step, the 5′ overhang is filled, copying the random barcode and generating blunt ends for <t>lambda</t> exonuclease digestion. 4) Lambda exonuclease (blue Pacman) digests until it encounters a physical barrier such as a cross-linked protein-DNA complex (‘Do not enter’ sign = ‘stop base’). 5) Single-stranded DNA is eluted and purified. 6) Self-circularization places the barcode next to the ‘stop base’. 7) An oligonucleotide (red arc) is paired with the region around the BamHI site for BamHI digestion (black scissors). 8) The digestion results in re-linearized DNA fragments with suitable Illumina sequences on both ends, ready for PCR library amplification. 9) Using single-end sequencing with the standard Illumina primer, each fragment is sequenced: first the barcode, then the genomic sequence starting with the ‘stop base’. 10) After alignment of the genomic sequences, reads with identical start positions and identical barcodes are removed. The final output is the position, number and strand orientation of the ‘stop’ bases. The frequencies of ‘stop’ bases on the positive strand are shown in red, while those on the negative strand are shown in blue. (b–e) Comparison of conventional ChIP-seq data (extended reads), ChIP-nexus data (raw stop base reads) and data generated using the original ChIP-exo protocol (raw stop base reads). (b) TBP profiles in human K562 cells at the RPS12 promoter. Although ChIP-nexus and ChIP-exo generally agree on TBP binding footprints, ChIP-nexus provides better coverage and richer details than ChIP-exo, which shows signs of over-amplification as large numbers of reads accumulate at a few discreet bases. (c) Dorsal profiles at the D. melanogaster decapentaplegic (dpp) enhancer. Five “Strong” dorsal binding sites (S1–S5) were previously mapped by in vitro DNase footprinting 12 . Note that ChIP-nexus identifies S4 as the only site with significant Dorsal binding in vivo . At the same time, ChIP-exo performed by Peconic did not detect any clear Dorsal footprint within the enhancer, in part due to the low read counts obtained. (d) Dorsal profiles at the rhomboid (rho) NEE enhancer. Four Dorsal binding sites (d1–d4) were previously mapped by in vitro DNase footprinting 14 . Note that ChIP-nexus identifies d3 as the strongest dorsal binding site in vivo , consistent with its close proximity to two Twist binding sites. Again, the original ChIP-exo protocol did not detect any clear Dorsal footprint within the enhancer. (e) Twist profiles at the same rho enhancer. Note that ChIP-nexus shows strong Twist footprints surrounding the two Twist binding sites (t1, t2) 14 . In this case, ChIP-exo performed by Peconic identified a similar Twist footprint. This shows that the Peconic experiments, which were performed with the same chromatin extracts as the Dorsal experiments, worked in principle but were less robust than our ChIP-nexus experiments.
    Lambda Exonuclease Digestion, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 76 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dna exonuclease digestions
    Superior performance of ChIP-nexus in discovering relevant binding footprints for transcription factors (a) Outline of ChIP-nexus 1) The transcription factor of interest (brown) is immunoprecipitated from chromatin fragments with antibodies in the same way as during conventional ChIP-seq experiments. 2) While still bound to the antibodies, the DNA ends are repaired, dA-tailed and then ligated to a special adaptor that contains a pair of sequences for library amplification (arrows indicate the correct orientation for them to be functional), a BamHI site (black dot) for linearization, and a 9-nucleotide barcode containing 5 random bases and 4 fixed bases to remove reads resulting from over-amplification of library DNA. The barcode is part of a 5′ overhang, which reduces adaptor-adaptor ligation. 3) After the adaptor ligation step, the 5′ overhang is filled, copying the random barcode and generating blunt ends for <t>lambda</t> exonuclease digestion. 4) Lambda exonuclease (blue Pacman) digests until it encounters a physical barrier such as a cross-linked protein-DNA complex (‘Do not enter’ sign = ‘stop base’). 5) Single-stranded DNA is eluted and purified. 6) Self-circularization places the barcode next to the ‘stop base’. 7) An oligonucleotide (red arc) is paired with the region around the BamHI site for BamHI digestion (black scissors). 8) The digestion results in re-linearized DNA fragments with suitable Illumina sequences on both ends, ready for PCR library amplification. 9) Using single-end sequencing with the standard Illumina primer, each fragment is sequenced: first the barcode, then the genomic sequence starting with the ‘stop base’. 10) After alignment of the genomic sequences, reads with identical start positions and identical barcodes are removed. The final output is the position, number and strand orientation of the ‘stop’ bases. The frequencies of ‘stop’ bases on the positive strand are shown in red, while those on the negative strand are shown in blue. (b–e) Comparison of conventional ChIP-seq data (extended reads), ChIP-nexus data (raw stop base reads) and data generated using the original ChIP-exo protocol (raw stop base reads). (b) TBP profiles in human K562 cells at the RPS12 promoter. Although ChIP-nexus and ChIP-exo generally agree on TBP binding footprints, ChIP-nexus provides better coverage and richer details than ChIP-exo, which shows signs of over-amplification as large numbers of reads accumulate at a few discreet bases. (c) Dorsal profiles at the D. melanogaster decapentaplegic (dpp) enhancer. Five “Strong” dorsal binding sites (S1–S5) were previously mapped by in vitro DNase footprinting 12 . Note that ChIP-nexus identifies S4 as the only site with significant Dorsal binding in vivo . At the same time, ChIP-exo performed by Peconic did not detect any clear Dorsal footprint within the enhancer, in part due to the low read counts obtained. (d) Dorsal profiles at the rhomboid (rho) NEE enhancer. Four Dorsal binding sites (d1–d4) were previously mapped by in vitro DNase footprinting 14 . Note that ChIP-nexus identifies d3 as the strongest dorsal binding site in vivo , consistent with its close proximity to two Twist binding sites. Again, the original ChIP-exo protocol did not detect any clear Dorsal footprint within the enhancer. (e) Twist profiles at the same rho enhancer. Note that ChIP-nexus shows strong Twist footprints surrounding the two Twist binding sites (t1, t2) 14 . In this case, ChIP-exo performed by Peconic identified a similar Twist footprint. This shows that the Peconic experiments, which were performed with the same chromatin extracts as the Dorsal experiments, worked in principle but were less robust than our ChIP-nexus experiments.
    Dna Exonuclease Digestions, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    TaKaRa exonuclease digestion
    The distal region of the IL-6 promoter contains functional C/EBP motifs A. IL-6 promoter Luciferase activity in PC3 cells transfected with the <t>pGL2-IL-6</t> deletion mutants shown on the left and pRV with and without treatment with rGal-3BP (2.5 µg/ml) for 24 hours. The data represent the mean (±SD) ratio Firefly Luciferase/Renilla Luciferase and are representative of one among <t>three</t> separate experiments each performed in triplicate samples. B. Sequencing analysis of the region of the IL-6 promoter extending from position −1586 to −1255 showing the presence of 3 C/EBP domains. C. EMSA of nuclear extracts from PC3 cells treated with rGAL-3BP (5 µg/ml) using biotinylated oligonucleotides in the presence of 10× fold excess of non-biotinylated oligonucleotides when indicated.
    Exonuclease Digestion, supplied by TaKaRa, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore exonuclease digestion
    The distal region of the IL-6 promoter contains functional C/EBP motifs A. IL-6 promoter Luciferase activity in PC3 cells transfected with the <t>pGL2-IL-6</t> deletion mutants shown on the left and pRV with and without treatment with rGal-3BP (2.5 µg/ml) for 24 hours. The data represent the mean (±SD) ratio Firefly Luciferase/Renilla Luciferase and are representative of one among <t>three</t> separate experiments each performed in triplicate samples. B. Sequencing analysis of the region of the IL-6 promoter extending from position −1586 to −1255 showing the presence of 3 C/EBP domains. C. EMSA of nuclear extracts from PC3 cells treated with rGAL-3BP (5 µg/ml) using biotinylated oligonucleotides in the presence of 10× fold excess of non-biotinylated oligonucleotides when indicated.
    Exonuclease Digestion, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Promega exonuclease iii digestion
    Repression of <t>HIF-1β</t> promoter activity by IFN-γ. A , Cloned HIF-1β promoter and series of truncated promoter-luciferase reporter constructs generated using exonuclease <t>III</t> digestion. Relative positions of each clone and major TSSs
    Exonuclease Iii Digestion, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 95 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Boehringer Mannheim λ exonuclease digestion
    Repression of <t>HIF-1β</t> promoter activity by IFN-γ. A , Cloned HIF-1β promoter and series of truncated promoter-luciferase reporter constructs generated using exonuclease <t>III</t> digestion. Relative positions of each clone and major TSSs
    λ Exonuclease Digestion, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare exonuclease i digestion
    Repression of <t>HIF-1β</t> promoter activity by IFN-γ. A , Cloned HIF-1β promoter and series of truncated promoter-luciferase reporter constructs generated using exonuclease <t>III</t> digestion. Relative positions of each clone and major TSSs
    Exonuclease I Digestion, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 88/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Pacific Biosciences exonuclease digestion
    Repression of <t>HIF-1β</t> promoter activity by IFN-γ. A , Cloned HIF-1β promoter and series of truncated promoter-luciferase reporter constructs generated using exonuclease <t>III</t> digestion. Relative positions of each clone and major TSSs
    Exonuclease Digestion, supplied by Pacific Biosciences, used in various techniques. Bioz Stars score: 89/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs t5 exonuclease digestion
    Repression of <t>HIF-1β</t> promoter activity by IFN-γ. A , Cloned HIF-1β promoter and series of truncated promoter-luciferase reporter constructs generated using exonuclease <t>III</t> digestion. Relative positions of each clone and major TSSs
    T5 Exonuclease Digestion, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    New England Biolabs t7 exonuclease digestion
    <t>T7</t> exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.
    T7 Exonuclease Digestion, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher λ exonuclease fermentas digestion
    <t>T7</t> exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.
    λ Exonuclease Fermentas Digestion, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs exonuclease digestion
    <t>T7</t> exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.
    Exonuclease Digestion, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher exonuclease thermoscientific digestion
    <t>T7</t> exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.
    Exonuclease Thermoscientific Digestion, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs exonuclease iii digestion
    <t>T7</t> exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.
    Exonuclease Iii Digestion, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Superior performance of ChIP-nexus in discovering relevant binding footprints for transcription factors (a) Outline of ChIP-nexus 1) The transcription factor of interest (brown) is immunoprecipitated from chromatin fragments with antibodies in the same way as during conventional ChIP-seq experiments. 2) While still bound to the antibodies, the DNA ends are repaired, dA-tailed and then ligated to a special adaptor that contains a pair of sequences for library amplification (arrows indicate the correct orientation for them to be functional), a BamHI site (black dot) for linearization, and a 9-nucleotide barcode containing 5 random bases and 4 fixed bases to remove reads resulting from over-amplification of library DNA. The barcode is part of a 5′ overhang, which reduces adaptor-adaptor ligation. 3) After the adaptor ligation step, the 5′ overhang is filled, copying the random barcode and generating blunt ends for lambda exonuclease digestion. 4) Lambda exonuclease (blue Pacman) digests until it encounters a physical barrier such as a cross-linked protein-DNA complex (‘Do not enter’ sign = ‘stop base’). 5) Single-stranded DNA is eluted and purified. 6) Self-circularization places the barcode next to the ‘stop base’. 7) An oligonucleotide (red arc) is paired with the region around the BamHI site for BamHI digestion (black scissors). 8) The digestion results in re-linearized DNA fragments with suitable Illumina sequences on both ends, ready for PCR library amplification. 9) Using single-end sequencing with the standard Illumina primer, each fragment is sequenced: first the barcode, then the genomic sequence starting with the ‘stop base’. 10) After alignment of the genomic sequences, reads with identical start positions and identical barcodes are removed. The final output is the position, number and strand orientation of the ‘stop’ bases. The frequencies of ‘stop’ bases on the positive strand are shown in red, while those on the negative strand are shown in blue. (b–e) Comparison of conventional ChIP-seq data (extended reads), ChIP-nexus data (raw stop base reads) and data generated using the original ChIP-exo protocol (raw stop base reads). (b) TBP profiles in human K562 cells at the RPS12 promoter. Although ChIP-nexus and ChIP-exo generally agree on TBP binding footprints, ChIP-nexus provides better coverage and richer details than ChIP-exo, which shows signs of over-amplification as large numbers of reads accumulate at a few discreet bases. (c) Dorsal profiles at the D. melanogaster decapentaplegic (dpp) enhancer. Five “Strong” dorsal binding sites (S1–S5) were previously mapped by in vitro DNase footprinting 12 . Note that ChIP-nexus identifies S4 as the only site with significant Dorsal binding in vivo . At the same time, ChIP-exo performed by Peconic did not detect any clear Dorsal footprint within the enhancer, in part due to the low read counts obtained. (d) Dorsal profiles at the rhomboid (rho) NEE enhancer. Four Dorsal binding sites (d1–d4) were previously mapped by in vitro DNase footprinting 14 . Note that ChIP-nexus identifies d3 as the strongest dorsal binding site in vivo , consistent with its close proximity to two Twist binding sites. Again, the original ChIP-exo protocol did not detect any clear Dorsal footprint within the enhancer. (e) Twist profiles at the same rho enhancer. Note that ChIP-nexus shows strong Twist footprints surrounding the two Twist binding sites (t1, t2) 14 . In this case, ChIP-exo performed by Peconic identified a similar Twist footprint. This shows that the Peconic experiments, which were performed with the same chromatin extracts as the Dorsal experiments, worked in principle but were less robust than our ChIP-nexus experiments.

    Journal: Nature biotechnology

    Article Title: ChIP-nexus: a novel ChIP-exo protocol for improved detection of in vivo transcription factor binding footprints

    doi: 10.1038/nbt.3121

    Figure Lengend Snippet: Superior performance of ChIP-nexus in discovering relevant binding footprints for transcription factors (a) Outline of ChIP-nexus 1) The transcription factor of interest (brown) is immunoprecipitated from chromatin fragments with antibodies in the same way as during conventional ChIP-seq experiments. 2) While still bound to the antibodies, the DNA ends are repaired, dA-tailed and then ligated to a special adaptor that contains a pair of sequences for library amplification (arrows indicate the correct orientation for them to be functional), a BamHI site (black dot) for linearization, and a 9-nucleotide barcode containing 5 random bases and 4 fixed bases to remove reads resulting from over-amplification of library DNA. The barcode is part of a 5′ overhang, which reduces adaptor-adaptor ligation. 3) After the adaptor ligation step, the 5′ overhang is filled, copying the random barcode and generating blunt ends for lambda exonuclease digestion. 4) Lambda exonuclease (blue Pacman) digests until it encounters a physical barrier such as a cross-linked protein-DNA complex (‘Do not enter’ sign = ‘stop base’). 5) Single-stranded DNA is eluted and purified. 6) Self-circularization places the barcode next to the ‘stop base’. 7) An oligonucleotide (red arc) is paired with the region around the BamHI site for BamHI digestion (black scissors). 8) The digestion results in re-linearized DNA fragments with suitable Illumina sequences on both ends, ready for PCR library amplification. 9) Using single-end sequencing with the standard Illumina primer, each fragment is sequenced: first the barcode, then the genomic sequence starting with the ‘stop base’. 10) After alignment of the genomic sequences, reads with identical start positions and identical barcodes are removed. The final output is the position, number and strand orientation of the ‘stop’ bases. The frequencies of ‘stop’ bases on the positive strand are shown in red, while those on the negative strand are shown in blue. (b–e) Comparison of conventional ChIP-seq data (extended reads), ChIP-nexus data (raw stop base reads) and data generated using the original ChIP-exo protocol (raw stop base reads). (b) TBP profiles in human K562 cells at the RPS12 promoter. Although ChIP-nexus and ChIP-exo generally agree on TBP binding footprints, ChIP-nexus provides better coverage and richer details than ChIP-exo, which shows signs of over-amplification as large numbers of reads accumulate at a few discreet bases. (c) Dorsal profiles at the D. melanogaster decapentaplegic (dpp) enhancer. Five “Strong” dorsal binding sites (S1–S5) were previously mapped by in vitro DNase footprinting 12 . Note that ChIP-nexus identifies S4 as the only site with significant Dorsal binding in vivo . At the same time, ChIP-exo performed by Peconic did not detect any clear Dorsal footprint within the enhancer, in part due to the low read counts obtained. (d) Dorsal profiles at the rhomboid (rho) NEE enhancer. Four Dorsal binding sites (d1–d4) were previously mapped by in vitro DNase footprinting 14 . Note that ChIP-nexus identifies d3 as the strongest dorsal binding site in vivo , consistent with its close proximity to two Twist binding sites. Again, the original ChIP-exo protocol did not detect any clear Dorsal footprint within the enhancer. (e) Twist profiles at the same rho enhancer. Note that ChIP-nexus shows strong Twist footprints surrounding the two Twist binding sites (t1, t2) 14 . In this case, ChIP-exo performed by Peconic identified a similar Twist footprint. This shows that the Peconic experiments, which were performed with the same chromatin extracts as the Dorsal experiments, worked in principle but were less robust than our ChIP-nexus experiments.

    Article Snippet: For lambda exonuclease digestion, each sample was incubated in 0.2 u/μl lambda exonuclease (New England Biolabs, M0262), 5% DMSO and 0.1% triton X-100 in 100 μl 1x NEB Lambda exonuclease reaction buffer at 37 °C for 60 min with constant agitation, followed by washing steps as above.

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Amplification, Functional Assay, Ligation, Purification, Polymerase Chain Reaction, Sequencing, Genomic Sequencing, Generated, In Vitro, Footprinting, In Vivo

    Analysis of the Dorsal, Twist and Max in vivo footprint (a–c) For each factor, the top 200 motifs with the highest ChIP-nexus read counts were selected and are shown in descending order as heat map. The footprints show a consistent boundary on the positive strand (red) and negative strand (blue) around each motif. The zoomed-in average profile below reveals that the footprints are wider than the motif. A schematic representation of the digestion pattern is shown below using Pacman symbols for lambda exonuclease. (a) The ChIP-nexus footprint for Dorsal (NFkB) on its canonical motif (GGRWWTTCC with up to one mismatch) extends on average 5 bp away from the motif edge. Thus, the average dorsal footprint is 18 bp long (horizontal black bar). (b) The Twist ChIP-nexus footprint on the E-box motif CABATG (no mismatch) has two outside boundaries, one at 11 bp, and one at 2 bp away from the motif edge, suggesting interactions with flanking DNA sequences. Each portion of the footprint is around 8–9bp long (horizontal black bar). (c) The Max ChIP-nexus footprint on its canonical E-box motif (CACGTG, no mismatch) has an outside boundary at 8 bp away from the motif edge, as well as a boundary inside the motif (at the A/T base), suggesting two partial footprints (horizontal black bars). (d, e) Average Max and Twist ChIP-nexus footprints at the top 200 sites for all possible E-box variants (CANNTG). Each variant profile includes its reverse complement. (d) Max binds specifically to the canonical CACGTG motif and to a lesser extent to the CACATG motif. Note that the Max footprint shape looks identical between the two motifs. (e) In contrast, the Twist binding specificity and the footprint shape is more complex. Notably, the outer boundary at -11bp is stronger at the CATATG and CACATG motif, whereas the inner boundary at -2 bp is stronger at the CAGATG motif.

    Journal: Nature biotechnology

    Article Title: ChIP-nexus: a novel ChIP-exo protocol for improved detection of in vivo transcription factor binding footprints

    doi: 10.1038/nbt.3121

    Figure Lengend Snippet: Analysis of the Dorsal, Twist and Max in vivo footprint (a–c) For each factor, the top 200 motifs with the highest ChIP-nexus read counts were selected and are shown in descending order as heat map. The footprints show a consistent boundary on the positive strand (red) and negative strand (blue) around each motif. The zoomed-in average profile below reveals that the footprints are wider than the motif. A schematic representation of the digestion pattern is shown below using Pacman symbols for lambda exonuclease. (a) The ChIP-nexus footprint for Dorsal (NFkB) on its canonical motif (GGRWWTTCC with up to one mismatch) extends on average 5 bp away from the motif edge. Thus, the average dorsal footprint is 18 bp long (horizontal black bar). (b) The Twist ChIP-nexus footprint on the E-box motif CABATG (no mismatch) has two outside boundaries, one at 11 bp, and one at 2 bp away from the motif edge, suggesting interactions with flanking DNA sequences. Each portion of the footprint is around 8–9bp long (horizontal black bar). (c) The Max ChIP-nexus footprint on its canonical E-box motif (CACGTG, no mismatch) has an outside boundary at 8 bp away from the motif edge, as well as a boundary inside the motif (at the A/T base), suggesting two partial footprints (horizontal black bars). (d, e) Average Max and Twist ChIP-nexus footprints at the top 200 sites for all possible E-box variants (CANNTG). Each variant profile includes its reverse complement. (d) Max binds specifically to the canonical CACGTG motif and to a lesser extent to the CACATG motif. Note that the Max footprint shape looks identical between the two motifs. (e) In contrast, the Twist binding specificity and the footprint shape is more complex. Notably, the outer boundary at -11bp is stronger at the CATATG and CACATG motif, whereas the inner boundary at -2 bp is stronger at the CAGATG motif.

    Article Snippet: For lambda exonuclease digestion, each sample was incubated in 0.2 u/μl lambda exonuclease (New England Biolabs, M0262), 5% DMSO and 0.1% triton X-100 in 100 μl 1x NEB Lambda exonuclease reaction buffer at 37 °C for 60 min with constant agitation, followed by washing steps as above.

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

    In situ detection of DNA using padlock probes and target primed rolling circle DNA synthesis . (A) The samples are cleaved with a restriction enzyme having a restriction site positioned 3' to the probe binding sequence. It is important that the enzyme does not have any other cleavage sites in close proximity to the 5'-end of the probe binding sequence to avoid degradation of the recognition sequence during exonuclease treatment. (B) The target sequence is made single stranded using the lambda exonuclease which digests duplex DNA in the 5'→3' direction in a highly processive manner, thereby making the target sequence single stranded. (C) The padlock probe is hybridized and ligated on the target sequence. Only padlock probes which are correctly hybridized at the point of ligation will be circularized. (D-E) The rolling circle reaction is initiated by using the target sequence as a primer, thereby locking the rolling circle product to the target sequence. (F) The rolling circle product is visualized by hybridizing a labeled oligonucleotide to the part of the padlock probe not recognizing the genomic hybridization target.

    Journal: BMC Molecular Biology

    Article Title: Detection of short repeated genomic sequences on metaphase chromosomes using padlock probes and target primed rolling circle DNA synthesis

    doi: 10.1186/1471-2199-8-103

    Figure Lengend Snippet: In situ detection of DNA using padlock probes and target primed rolling circle DNA synthesis . (A) The samples are cleaved with a restriction enzyme having a restriction site positioned 3' to the probe binding sequence. It is important that the enzyme does not have any other cleavage sites in close proximity to the 5'-end of the probe binding sequence to avoid degradation of the recognition sequence during exonuclease treatment. (B) The target sequence is made single stranded using the lambda exonuclease which digests duplex DNA in the 5'→3' direction in a highly processive manner, thereby making the target sequence single stranded. (C) The padlock probe is hybridized and ligated on the target sequence. Only padlock probes which are correctly hybridized at the point of ligation will be circularized. (D-E) The rolling circle reaction is initiated by using the target sequence as a primer, thereby locking the rolling circle product to the target sequence. (F) The rolling circle product is visualized by hybridizing a labeled oligonucleotide to the part of the padlock probe not recognizing the genomic hybridization target.

    Article Snippet: Lambda exonuclease Exonuclease digestion was performed in a buffer containing 1× lambda exonuclease buffer (NEB), 0.2 μg/μl BSA (NEB) and 1u/μl lambda exonuclease (NEB) for 1 min at 37°C.

    Techniques: In Situ, DNA Synthesis, Binding Assay, Sequencing, Ligation, Labeling, Hybridization

    ChIP-exo 5.0 increases library yield. a Schematic of ChIP-exo 5.0. The purple triangle indicates the location of the Read_1 start site, which is also the λ exonuclease stop site. b 2% agarose gel of the electrophoresed library following 18 cycles of PCR for various S. cerevisiae transcription factors assayed by ChIP-exo 1.1 or 5.0. Following ChIP, the sample was split and libraries prepared using the indicated protocols. After splitting the sample, each reaction contained a 50 ml cell equivalent (OD 600 = 0.8) of yeast chromatin, which is five-fold less than the amount optimized for ChIP-exo 1.1. ChIP-exo 5.0 produced greater library yield for all samples. c Heatmaps comparing ChIP-exo 1.1 and 5.0 at the 975 Reb1 primary motifs in a 200 bp window. d Composite plot of data from panel ( c )

    Journal: Nature Communications

    Article Title: Simplified ChIP-exo assays

    doi: 10.1038/s41467-018-05265-7

    Figure Lengend Snippet: ChIP-exo 5.0 increases library yield. a Schematic of ChIP-exo 5.0. The purple triangle indicates the location of the Read_1 start site, which is also the λ exonuclease stop site. b 2% agarose gel of the electrophoresed library following 18 cycles of PCR for various S. cerevisiae transcription factors assayed by ChIP-exo 1.1 or 5.0. Following ChIP, the sample was split and libraries prepared using the indicated protocols. After splitting the sample, each reaction contained a 50 ml cell equivalent (OD 600 = 0.8) of yeast chromatin, which is five-fold less than the amount optimized for ChIP-exo 1.1. ChIP-exo 5.0 produced greater library yield for all samples. c Heatmaps comparing ChIP-exo 1.1 and 5.0 at the 975 Reb1 primary motifs in a 200 bp window. d Composite plot of data from panel ( c )

    Article Snippet: The λ exonuclease digestion (100 µl) containing: 20 U λ exonuclease (NEB), 1 × λ exonuclease reaction buffer (NEB), 0.1% Triton-X 100, and 5% DMSO was incubated for 30 min at 37 °C; then washed with 10 mM Tris-HCl, pH 8.0 at 4 °C.

    Techniques: Chromatin Immunoprecipitation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Produced

    The distal region of the IL-6 promoter contains functional C/EBP motifs A. IL-6 promoter Luciferase activity in PC3 cells transfected with the pGL2-IL-6 deletion mutants shown on the left and pRV with and without treatment with rGal-3BP (2.5 µg/ml) for 24 hours. The data represent the mean (±SD) ratio Firefly Luciferase/Renilla Luciferase and are representative of one among three separate experiments each performed in triplicate samples. B. Sequencing analysis of the region of the IL-6 promoter extending from position −1586 to −1255 showing the presence of 3 C/EBP domains. C. EMSA of nuclear extracts from PC3 cells treated with rGAL-3BP (5 µg/ml) using biotinylated oligonucleotides in the presence of 10× fold excess of non-biotinylated oligonucleotides when indicated.

    Journal: Cancer research

    Article Title: A GALECTIN-3-DEPENDENT PATHWAY UPREGULATES INTERLEUKIN-6 IN THE MICROENVIRONMENT OF HUMAN NEUROBLASTOMA

    doi: 10.1158/0008-5472.CAN-11-2165

    Figure Lengend Snippet: The distal region of the IL-6 promoter contains functional C/EBP motifs A. IL-6 promoter Luciferase activity in PC3 cells transfected with the pGL2-IL-6 deletion mutants shown on the left and pRV with and without treatment with rGal-3BP (2.5 µg/ml) for 24 hours. The data represent the mean (±SD) ratio Firefly Luciferase/Renilla Luciferase and are representative of one among three separate experiments each performed in triplicate samples. B. Sequencing analysis of the region of the IL-6 promoter extending from position −1586 to −1255 showing the presence of 3 C/EBP domains. C. EMSA of nuclear extracts from PC3 cells treated with rGAL-3BP (5 µg/ml) using biotinylated oligonucleotides in the presence of 10× fold excess of non-biotinylated oligonucleotides when indicated.

    Article Snippet: IL-6 promoter deletion mutants in the pGL2-IL-6-Luc construct were generated either by Exonuclease III digestion using the Deletion Kit for kilo sequencing (Takara) in accordance with the instructions of the manufacturer (deletion −1041) or by restriction endonuclease digestion with KpnI and NheI (deletion mutant −212) followed by Klenow fragment reaction at 37°C for 15 minutes.

    Techniques: Functional Assay, Luciferase, Activity Assay, Transfection, Sequencing

    Repression of HIF-1β promoter activity by IFN-γ. A , Cloned HIF-1β promoter and series of truncated promoter-luciferase reporter constructs generated using exonuclease III digestion. Relative positions of each clone and major TSSs

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: IFN-? Attenuates Hypoxia-Inducible Factor (HIF) Activity in Intestinal Epithelial Cells through Transcriptional Repression of HIF-1?

    doi: 10.4049/jimmunol.1001442

    Figure Lengend Snippet: Repression of HIF-1β promoter activity by IFN-γ. A , Cloned HIF-1β promoter and series of truncated promoter-luciferase reporter constructs generated using exonuclease III digestion. Relative positions of each clone and major TSSs

    Article Snippet: Sequential truncations of the cloned HIF-1β promoter sequence were generated by exonuclease III digestion (Erase-a-Base; Promega).

    Techniques: Activity Assay, Clone Assay, Luciferase, Construct, Generated

    T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.

    Journal: The Journal of Biological Chemistry

    Article Title: Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *

    doi: 10.1074/jbc.M110.140350

    Figure Lengend Snippet: T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.

    Article Snippet: T7 exonuclease digestion was performed by incubating purified EJ products with either increasing concentrations or 5 units of T7 exonuclease (New England Biolabs) at 25 °C for 2 h. In some cases, a fraction of EJ products was digested with XhoI (4 units) (37 °C for 4 h) prior to T7 exonuclease digestion.

    Techniques: Incubation, Polyacrylamide Gel Electrophoresis