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Application of PAtCh-Cap to <t>CTCF</t> <t>ChIP-exo</t> data allowed for significant artifact removal and improved confidence in peak identification. ( A ) To identify high-confidence CTCF peaks, all peaks called with a 0.05 q -value threshold from the ChIP-exo data with (red) and without (black) input treatment were plotted as the –log(q-value) versus ranked peak number. High confidence peaks were determined to be those characterized by a –log( q -value) higher than the inflection point and a line with a slope of –tan 1 to the curve (denoted by vertical lines). ( B ) Venn diagram demonstrating the overlap of high-confidence peaks identified from data sets with and without input treatment (top). The number of CTCF motifs found within each pool is denoted and clearly shows that the percentage of CTCF containing peaks relative to the total increases substantially after input treatment. Venn diagrams for the overlap of blacklisted peaks with each of the above regions (bottom).
Chip Exo Kit, supplied by Active Motif, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond"

Article Title: PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw741

Application of PAtCh-Cap to CTCF ChIP-exo data allowed for significant artifact removal and improved confidence in peak identification. ( A ) To identify high-confidence CTCF peaks, all peaks called with a 0.05 q -value threshold from the ChIP-exo data with (red) and without (black) input treatment were plotted as the –log(q-value) versus ranked peak number. High confidence peaks were determined to be those characterized by a –log( q -value) higher than the inflection point and a line with a slope of –tan 1 to the curve (denoted by vertical lines). ( B ) Venn diagram demonstrating the overlap of high-confidence peaks identified from data sets with and without input treatment (top). The number of CTCF motifs found within each pool is denoted and clearly shows that the percentage of CTCF containing peaks relative to the total increases substantially after input treatment. Venn diagrams for the overlap of blacklisted peaks with each of the above regions (bottom).
Figure Legend Snippet: Application of PAtCh-Cap to CTCF ChIP-exo data allowed for significant artifact removal and improved confidence in peak identification. ( A ) To identify high-confidence CTCF peaks, all peaks called with a 0.05 q -value threshold from the ChIP-exo data with (red) and without (black) input treatment were plotted as the –log(q-value) versus ranked peak number. High confidence peaks were determined to be those characterized by a –log( q -value) higher than the inflection point and a line with a slope of –tan 1 to the curve (denoted by vertical lines). ( B ) Venn diagram demonstrating the overlap of high-confidence peaks identified from data sets with and without input treatment (top). The number of CTCF motifs found within each pool is denoted and clearly shows that the percentage of CTCF containing peaks relative to the total increases substantially after input treatment. Venn diagrams for the overlap of blacklisted peaks with each of the above regions (bottom).

Techniques Used: Chromatin Immunoprecipitation

Representative CTCF ChIP-exo read coverage tracks for the pericentromeric region of chromosome 1 ( A ) and the promoter of the KCNJ3 gene ( B ). The CTCF reads (blue) were normalized to the reads from the input control (green) using MACS2 ( 27 ) to generate the enrichment read coverage tracks (red). Peaks identified by the MACS2 peak caller (represented in the .bed tracks) are denoted as red or blue vertical lines for the CTCF ChIP-exo data sets with and without input treatment, respectively. Analysis of the genomic sequences underneath the remaining peaks after input treatment (vertical red lines) definitively showed that these sites contain the core CTCF motif as evidenced by alignment of the CTCF sequence logo beneath.
Figure Legend Snippet: Representative CTCF ChIP-exo read coverage tracks for the pericentromeric region of chromosome 1 ( A ) and the promoter of the KCNJ3 gene ( B ). The CTCF reads (blue) were normalized to the reads from the input control (green) using MACS2 ( 27 ) to generate the enrichment read coverage tracks (red). Peaks identified by the MACS2 peak caller (represented in the .bed tracks) are denoted as red or blue vertical lines for the CTCF ChIP-exo data sets with and without input treatment, respectively. Analysis of the genomic sequences underneath the remaining peaks after input treatment (vertical red lines) definitively showed that these sites contain the core CTCF motif as evidenced by alignment of the CTCF sequence logo beneath.

Techniques Used: Chromatin Immunoprecipitation, Genomic Sequencing, Sequencing

From the input treated CTCF ChIP-exo data set, ( A ) read tag distributions around all genomic CTCF-bound sites shown in the four binned motif combinations (right panel) were centered on the midpoint of the CTCF consensus to generate a heat map (top left) which is summed below as an aggregate plot. Denoted in blue and red are the sense and antisense strand read enrichments around the core CTCF motif, respectively. The centralized CTCF core sequence and adjacent motifs are depicted above a color map representation of 50 bp DNA stretches containing the various motif combinations (right panel). ( B ) Heat maps from RNA-seq data depicting gene transcripts exhibiting a two-fold up- (green) or down-regulation (red) after CTCF depletion relative to the scrambled siRNA control (Scr). For each motif group, CTCF promoter occupation sites (defined as ±1000 bps around the transcription start site (TSS)) were intersected with the RNA-seq data and resulting altered gene sets were binned as individual heat maps. ( C ) Each gene set from ( B ) was subjected to Ingenuity Pathway Analysis (IPA, www.ingenuity.com ) to identify biological pathways uniquely modulated by each of the CTCF motif combinations. ( D–F ) The same analyses in ( A–C ) were performed separately on the core CTCF consensus with the newly identified 3′-CTCF motif.
Figure Legend Snippet: From the input treated CTCF ChIP-exo data set, ( A ) read tag distributions around all genomic CTCF-bound sites shown in the four binned motif combinations (right panel) were centered on the midpoint of the CTCF consensus to generate a heat map (top left) which is summed below as an aggregate plot. Denoted in blue and red are the sense and antisense strand read enrichments around the core CTCF motif, respectively. The centralized CTCF core sequence and adjacent motifs are depicted above a color map representation of 50 bp DNA stretches containing the various motif combinations (right panel). ( B ) Heat maps from RNA-seq data depicting gene transcripts exhibiting a two-fold up- (green) or down-regulation (red) after CTCF depletion relative to the scrambled siRNA control (Scr). For each motif group, CTCF promoter occupation sites (defined as ±1000 bps around the transcription start site (TSS)) were intersected with the RNA-seq data and resulting altered gene sets were binned as individual heat maps. ( C ) Each gene set from ( B ) was subjected to Ingenuity Pathway Analysis (IPA, www.ingenuity.com ) to identify biological pathways uniquely modulated by each of the CTCF motif combinations. ( D–F ) The same analyses in ( A–C ) were performed separately on the core CTCF consensus with the newly identified 3′-CTCF motif.

Techniques Used: Chromatin Immunoprecipitation, Sequencing, RNA Sequencing Assay, Indirect Immunoperoxidase Assay

Related Articles

Centrifugation:

Article Title: PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond
Article Snippet: The IP, exonuclease digestions and library generation procedures were all performed using a commercially available ChIP-exo Kit (Active Motif) following the manufacturer's instructions with the few noted modifications. .. A Diagenode Bioruptor Standard sonication device (run at max amplitude for 5 × 15 min in ice water) was used to shear the cross-linked DNA to 100–400 bp fragments.

Article Title: PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond
Article Snippet: Cell debris was removed by centrifugation and the supernatant containing the solubilized chromatin DNA:protein complexes was isolated. .. It should be noted that the library preparations performed with the Active Motif ChIP-exo Kit are designed to be compatible with the Illumina sequencing platform ( ).

Sample Prep:

Article Title: PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond
Article Snippet: The IP, exonuclease digestions and library generation procedures were all performed using a commercially available ChIP-exo Kit (Active Motif) following the manufacturer's instructions with the few noted modifications. .. Cell debris was removed by centrifugation and the supernatant containing the solubilized chromatin DNA:protein complexes was isolated.

Article Title: PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond
Article Snippet: It should be noted that the library preparations performed with the Active Motif ChIP-exo Kit are designed to be compatible with the Illumina sequencing platform ( ). .. It should be noted that the library preparations performed with the Active Motif ChIP-exo Kit are designed to be compatible with the Illumina sequencing platform ( ).

Magnetic Beads:

Article Title: PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond
Article Snippet: For each of the two replicates, the input sample (obtained as discussed above) was combined with 300 μl of 0.1 M EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) along with the pre-functionalized biotinylated magnetics beads and incubated in 10 ml 0.1 mM MES buffer (pH 5.0) for 3 h at room temperature on a mechanical rotator. .. Once covalently bound to the magnetic beads, the input samples were treated identically as described above for the CTCF ChIP-exo samples utilizing reagents and materials from the Active Motif ChIP-exo Kit. .. For each of the three replicates, 20 × 106 HeLa cells were fixed with 1% formaldehyde for 15 min to cross-link protein:DNA complexes, followed by a quench with 125 mM glycine.

Article Title: PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond
Article Snippet: Following two series of washes with 0.01 M PBS (pH 7.4), 50 μg of M-280 streptavidin coated Dynabeads (Thermo Fisher Scientific; equivalent to the number of beads utilized per reaction in the Active Motif ChIP-exo Kit) were conjugated with 10 μl of 50 nM EZ-link amine-PEG3-Biotin (Thermo Fisher Scientific; PEG = polyethylene glycol) in 0.01 M PBS (pH 7.4) at room temperature for 20 min. .. These beads were selected as they have the same size and core material composition as the protein G coated magnetic beads utilized in the Active Motif ChIP-exo Kit. .. The biotinylated beads were then washed twice with 0.01 M PBS (pH 7.4) and once with 0.1 mM MES (pH 5.0) to remove any non-conjugated material.

Article Title: PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond
Article Snippet: The IP, exonuclease digestions and library generation procedures were all performed using a commercially available ChIP-exo Kit (Active Motif) following the manufacturer's instructions with the few noted modifications. .. The IP, exonuclease digestions and library generation procedures were all performed using a commercially available ChIP-exo Kit (Active Motif) following the manufacturer's instructions with the few noted modifications.

Article Title: PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond
Article Snippet: For the IP step, protein G coated magnetic beads were pre-functionalized with CTCF antibody (Millipore) prior to incubation with the sheared chromatin sample. .. It should be noted that the library preparations performed with the Active Motif ChIP-exo Kit are designed to be compatible with the Illumina sequencing platform ( ).

Isolation:

Article Title: PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond
Article Snippet: The IP, exonuclease digestions and library generation procedures were all performed using a commercially available ChIP-exo Kit (Active Motif) following the manufacturer's instructions with the few noted modifications. .. A Diagenode Bioruptor Standard sonication device (run at max amplitude for 5 × 15 min in ice water) was used to shear the cross-linked DNA to 100–400 bp fragments.

Article Title: PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond
Article Snippet: Cell debris was removed by centrifugation and the supernatant containing the solubilized chromatin DNA:protein complexes was isolated. .. It should be noted that the library preparations performed with the Active Motif ChIP-exo Kit are designed to be compatible with the Illumina sequencing platform ( ).

Purification:

Article Title: PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond
Article Snippet: DNA purification after reverse cross-linking was performed with the MinElute PCR Purification Kit (Qiagen). .. It should be noted that the library preparations performed with the Active Motif ChIP-exo Kit are designed to be compatible with the Illumina sequencing platform ( ).

Sequencing:

Article Title: PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond
Article Snippet: DNA purification after reverse cross-linking was performed with the MinElute PCR Purification Kit (Qiagen). .. It should be noted that the library preparations performed with the Active Motif ChIP-exo Kit are designed to be compatible with the Illumina sequencing platform ( ). .. Final purified DNA libraries were sequenced by the High-Throughput Genomics Core within the University of Utah Huntsman Cancer Institute using the Illumina HiSeq 2000 platform.

Incubation:

Article Title: PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond
Article Snippet: For each of the two replicates, the input sample (obtained as discussed above) was combined with 300 μl of 0.1 M EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) along with the pre-functionalized biotinylated magnetics beads and incubated in 10 ml 0.1 mM MES buffer (pH 5.0) for 3 h at room temperature on a mechanical rotator. .. Once covalently bound to the magnetic beads, the input samples were treated identically as described above for the CTCF ChIP-exo samples utilizing reagents and materials from the Active Motif ChIP-exo Kit.

Article Title: PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond
Article Snippet: These beads were selected as they have the same size and core material composition as the protein G coated magnetic beads utilized in the Active Motif ChIP-exo Kit. .. These beads were selected as they have the same size and core material composition as the protein G coated magnetic beads utilized in the Active Motif ChIP-exo Kit.

Article Title: PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond
Article Snippet: The IP, exonuclease digestions and library generation procedures were all performed using a commercially available ChIP-exo Kit (Active Motif) following the manufacturer's instructions with the few noted modifications. .. The IP, exonuclease digestions and library generation procedures were all performed using a commercially available ChIP-exo Kit (Active Motif) following the manufacturer's instructions with the few noted modifications.

Article Title: PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond
Article Snippet: For the IP step, protein G coated magnetic beads were pre-functionalized with CTCF antibody (Millipore) prior to incubation with the sheared chromatin sample. .. It should be noted that the library preparations performed with the Active Motif ChIP-exo Kit are designed to be compatible with the Illumina sequencing platform ( ).

DNA Purification:

Article Title: PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond
Article Snippet: DNA purification after reverse cross-linking was performed with the MinElute PCR Purification Kit (Qiagen). .. It should be noted that the library preparations performed with the Active Motif ChIP-exo Kit are designed to be compatible with the Illumina sequencing platform ( ).

Polymerase Chain Reaction:

Article Title: PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond
Article Snippet: DNA purification after reverse cross-linking was performed with the MinElute PCR Purification Kit (Qiagen). .. It should be noted that the library preparations performed with the Active Motif ChIP-exo Kit are designed to be compatible with the Illumina sequencing platform ( ).

Sonication:

Article Title: PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond
Article Snippet: A Diagenode Bioruptor Standard sonication device (run at max amplitude for 5 × 15 min in ice water) was used to shear the cross-linked DNA to 100–400 bp fragments. .. It should be noted that the library preparations performed with the Active Motif ChIP-exo Kit are designed to be compatible with the Illumina sequencing platform ( ).

Chromatin Immunoprecipitation:

Article Title: PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond
Article Snippet: For each of the two replicates, the input sample (obtained as discussed above) was combined with 300 μl of 0.1 M EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) along with the pre-functionalized biotinylated magnetics beads and incubated in 10 ml 0.1 mM MES buffer (pH 5.0) for 3 h at room temperature on a mechanical rotator. .. Once covalently bound to the magnetic beads, the input samples were treated identically as described above for the CTCF ChIP-exo samples utilizing reagents and materials from the Active Motif ChIP-exo Kit. .. For each of the three replicates, 20 × 106 HeLa cells were fixed with 1% formaldehyde for 15 min to cross-link protein:DNA complexes, followed by a quench with 125 mM glycine.

Article Title: PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond
Article Snippet: Following two series of washes with 0.01 M PBS (pH 7.4), 50 μg of M-280 streptavidin coated Dynabeads (Thermo Fisher Scientific; equivalent to the number of beads utilized per reaction in the Active Motif ChIP-exo Kit) were conjugated with 10 μl of 50 nM EZ-link amine-PEG3-Biotin (Thermo Fisher Scientific; PEG = polyethylene glycol) in 0.01 M PBS (pH 7.4) at room temperature for 20 min. .. These beads were selected as they have the same size and core material composition as the protein G coated magnetic beads utilized in the Active Motif ChIP-exo Kit. .. The biotinylated beads were then washed twice with 0.01 M PBS (pH 7.4) and once with 0.1 mM MES (pH 5.0) to remove any non-conjugated material.

Article Title: PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond
Article Snippet: For each of the two ChIP-exo replicates, 20 × 106 HeLa cells were fixed with 1% formaldehyde for 15 min to cross-link protein:DNA complexes, followed by a quench with 125 mM glycine. .. The IP, exonuclease digestions and library generation procedures were all performed using a commercially available ChIP-exo Kit (Active Motif) following the manufacturer's instructions with the few noted modifications. .. A Diagenode Bioruptor Standard sonication device (run at max amplitude for 5 × 15 min in ice water) was used to shear the cross-linked DNA to 100–400 bp fragments.

Article Title: PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond
Article Snippet: DNA purification after reverse cross-linking was performed with the MinElute PCR Purification Kit (Qiagen). .. It should be noted that the library preparations performed with the Active Motif ChIP-exo Kit are designed to be compatible with the Illumina sequencing platform ( ). .. Final purified DNA libraries were sequenced by the High-Throughput Genomics Core within the University of Utah Huntsman Cancer Institute using the Illumina HiSeq 2000 platform.

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    Active Motif chip exo kit
    Application of PAtCh-Cap to <t>CTCF</t> <t>ChIP-exo</t> data allowed for significant artifact removal and improved confidence in peak identification. ( A ) To identify high-confidence CTCF peaks, all peaks called with a 0.05 q -value threshold from the ChIP-exo data with (red) and without (black) input treatment were plotted as the –log(q-value) versus ranked peak number. High confidence peaks were determined to be those characterized by a –log( q -value) higher than the inflection point and a line with a slope of –tan 1 to the curve (denoted by vertical lines). ( B ) Venn diagram demonstrating the overlap of high-confidence peaks identified from data sets with and without input treatment (top). The number of CTCF motifs found within each pool is denoted and clearly shows that the percentage of CTCF containing peaks relative to the total increases substantially after input treatment. Venn diagrams for the overlap of blacklisted peaks with each of the above regions (bottom).
    Chip Exo Kit, supplied by Active Motif, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/chip exo kit/product/Active Motif
    Average 86 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    chip exo kit - by Bioz Stars, 2020-01
    86/100 stars
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    Application of PAtCh-Cap to CTCF ChIP-exo data allowed for significant artifact removal and improved confidence in peak identification. ( A ) To identify high-confidence CTCF peaks, all peaks called with a 0.05 q -value threshold from the ChIP-exo data with (red) and without (black) input treatment were plotted as the –log(q-value) versus ranked peak number. High confidence peaks were determined to be those characterized by a –log( q -value) higher than the inflection point and a line with a slope of –tan 1 to the curve (denoted by vertical lines). ( B ) Venn diagram demonstrating the overlap of high-confidence peaks identified from data sets with and without input treatment (top). The number of CTCF motifs found within each pool is denoted and clearly shows that the percentage of CTCF containing peaks relative to the total increases substantially after input treatment. Venn diagrams for the overlap of blacklisted peaks with each of the above regions (bottom).

    Journal: Nucleic Acids Research

    Article Title: PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond

    doi: 10.1093/nar/gkw741

    Figure Lengend Snippet: Application of PAtCh-Cap to CTCF ChIP-exo data allowed for significant artifact removal and improved confidence in peak identification. ( A ) To identify high-confidence CTCF peaks, all peaks called with a 0.05 q -value threshold from the ChIP-exo data with (red) and without (black) input treatment were plotted as the –log(q-value) versus ranked peak number. High confidence peaks were determined to be those characterized by a –log( q -value) higher than the inflection point and a line with a slope of –tan 1 to the curve (denoted by vertical lines). ( B ) Venn diagram demonstrating the overlap of high-confidence peaks identified from data sets with and without input treatment (top). The number of CTCF motifs found within each pool is denoted and clearly shows that the percentage of CTCF containing peaks relative to the total increases substantially after input treatment. Venn diagrams for the overlap of blacklisted peaks with each of the above regions (bottom).

    Article Snippet: Once covalently bound to the magnetic beads, the input samples were treated identically as described above for the CTCF ChIP-exo samples utilizing reagents and materials from the Active Motif ChIP-exo Kit.

    Techniques: Chromatin Immunoprecipitation

    Representative CTCF ChIP-exo read coverage tracks for the pericentromeric region of chromosome 1 ( A ) and the promoter of the KCNJ3 gene ( B ). The CTCF reads (blue) were normalized to the reads from the input control (green) using MACS2 ( 27 ) to generate the enrichment read coverage tracks (red). Peaks identified by the MACS2 peak caller (represented in the .bed tracks) are denoted as red or blue vertical lines for the CTCF ChIP-exo data sets with and without input treatment, respectively. Analysis of the genomic sequences underneath the remaining peaks after input treatment (vertical red lines) definitively showed that these sites contain the core CTCF motif as evidenced by alignment of the CTCF sequence logo beneath.

    Journal: Nucleic Acids Research

    Article Title: PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond

    doi: 10.1093/nar/gkw741

    Figure Lengend Snippet: Representative CTCF ChIP-exo read coverage tracks for the pericentromeric region of chromosome 1 ( A ) and the promoter of the KCNJ3 gene ( B ). The CTCF reads (blue) were normalized to the reads from the input control (green) using MACS2 ( 27 ) to generate the enrichment read coverage tracks (red). Peaks identified by the MACS2 peak caller (represented in the .bed tracks) are denoted as red or blue vertical lines for the CTCF ChIP-exo data sets with and without input treatment, respectively. Analysis of the genomic sequences underneath the remaining peaks after input treatment (vertical red lines) definitively showed that these sites contain the core CTCF motif as evidenced by alignment of the CTCF sequence logo beneath.

    Article Snippet: Once covalently bound to the magnetic beads, the input samples were treated identically as described above for the CTCF ChIP-exo samples utilizing reagents and materials from the Active Motif ChIP-exo Kit.

    Techniques: Chromatin Immunoprecipitation, Genomic Sequencing, Sequencing

    From the input treated CTCF ChIP-exo data set, ( A ) read tag distributions around all genomic CTCF-bound sites shown in the four binned motif combinations (right panel) were centered on the midpoint of the CTCF consensus to generate a heat map (top left) which is summed below as an aggregate plot. Denoted in blue and red are the sense and antisense strand read enrichments around the core CTCF motif, respectively. The centralized CTCF core sequence and adjacent motifs are depicted above a color map representation of 50 bp DNA stretches containing the various motif combinations (right panel). ( B ) Heat maps from RNA-seq data depicting gene transcripts exhibiting a two-fold up- (green) or down-regulation (red) after CTCF depletion relative to the scrambled siRNA control (Scr). For each motif group, CTCF promoter occupation sites (defined as ±1000 bps around the transcription start site (TSS)) were intersected with the RNA-seq data and resulting altered gene sets were binned as individual heat maps. ( C ) Each gene set from ( B ) was subjected to Ingenuity Pathway Analysis (IPA, www.ingenuity.com ) to identify biological pathways uniquely modulated by each of the CTCF motif combinations. ( D–F ) The same analyses in ( A–C ) were performed separately on the core CTCF consensus with the newly identified 3′-CTCF motif.

    Journal: Nucleic Acids Research

    Article Title: PAtCh-Cap: input strategy for improving analysis of ChIP-exo data sets and beyond

    doi: 10.1093/nar/gkw741

    Figure Lengend Snippet: From the input treated CTCF ChIP-exo data set, ( A ) read tag distributions around all genomic CTCF-bound sites shown in the four binned motif combinations (right panel) were centered on the midpoint of the CTCF consensus to generate a heat map (top left) which is summed below as an aggregate plot. Denoted in blue and red are the sense and antisense strand read enrichments around the core CTCF motif, respectively. The centralized CTCF core sequence and adjacent motifs are depicted above a color map representation of 50 bp DNA stretches containing the various motif combinations (right panel). ( B ) Heat maps from RNA-seq data depicting gene transcripts exhibiting a two-fold up- (green) or down-regulation (red) after CTCF depletion relative to the scrambled siRNA control (Scr). For each motif group, CTCF promoter occupation sites (defined as ±1000 bps around the transcription start site (TSS)) were intersected with the RNA-seq data and resulting altered gene sets were binned as individual heat maps. ( C ) Each gene set from ( B ) was subjected to Ingenuity Pathway Analysis (IPA, www.ingenuity.com ) to identify biological pathways uniquely modulated by each of the CTCF motif combinations. ( D–F ) The same analyses in ( A–C ) were performed separately on the core CTCF consensus with the newly identified 3′-CTCF motif.

    Article Snippet: Once covalently bound to the magnetic beads, the input samples were treated identically as described above for the CTCF ChIP-exo samples utilizing reagents and materials from the Active Motif ChIP-exo Kit.

    Techniques: Chromatin Immunoprecipitation, Sequencing, RNA Sequencing Assay, Indirect Immunoperoxidase Assay