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  • 94
    Name:
    CTCF Antibody
    Description:
    CCCTC binding factor CTCF and its paralog the Brother of the Regulator of Imprinted Sites BORIS are highly conserved transcription factors that regulate transcriptional activation and repression insulator function and imprinting control regions ICRs 1 4 Although they have divergent amino and carboxy termini both proteins contain 11 conserved zinc finger domains that work in combination to bind the same DNA elements 1 CTCF is ubiquitously expressed and contributes to transcriptional regulation of cell growth regulated genes including c myc p19 ARF p16 INK4A BRCA1 p53 p27 E2F1 and TERT 1 CTCF also binds to and is required for the enhancer blocking activity of all known insulator elements and ICRs including the H19 IgF2 Prader Willi Angelman syndrome and Inactive X Specific Transcript XIST anti sense loci 5 7 CTCF DNA binding is sensitive to DNA methylation a mark that determines selection of the imprinted allele maternal vs paternal 1 The various functions of CTCF are regulated by at least two different post translational modifications Poly ADP ribosyl ation of CTCF is required for insulator function 8 Phosphorylation of Ser612 by protein kinase CK2 facilitates a switch of CTCF from a transcriptional repressor to an activator at the c myc promoter 9 CTCF mutations or deletions have been found in many breast prostate and Wilms tumors 10 11 Expression of BORIS is restricted to spermatocytes and is mutually exclusive of CTCF 3 In cells expressing BORIS promoters of X linked cancer testis antigens like MAGE 1A are demethylated and activated but methylated and inactive in CTCF expressing somatic cells 12 Like other testis specific proteins BORIS is abnormally expressed in different cancers such as breast cancer and has a greater affinity than CTCF for DNA binding sites detracting from CTCF s potential tumor suppressing activity 1 3 13 14
    Catalog Number:
    2899
    Price:
    None
    Category:
    Primary Antibodies
    Source:
    Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the carboxy terminus of the human CTCF protein. Antibodies are purified by protein A and peptide affinity chromatography.
    Reactivity:
    Human Mouse Rat Monkey
    Applications:
    Western Blot, Immunoprecipitation, Immunofluorescence, Chromatin Immunoprecipitation
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc anti ctcf
    Topology of the EGFR locus in unamplified and amplified glioblastoma. ( a ) (Top) HiC contact map of unamplified glioblastoma model cell line G567. (Middle) <t>CTCF</t> ChlP-seq track of unamplified glioblastoma model cell line GSC23. The red and green arrows denote the orientation of the CTCF motif. (Bottom) Schematic of the boundaries of the loop domain. ( b ) 4C-seq profiles anchored from near the EGFR transcription start site (dotted line) showing interactions to the EGFR enhancers (boxed) for GSC23 (unamplified). Red arcs denote regions with strong distal interactions with EGFR at a 12 kb window thresholded at a 0.12 interaction frequency. <t>H3K27ac</t> ChIP-seq tracks accompany the 4C-seq data. The 4C-seq tracks consist of a main trend of the chromatin interactions based on a 5 kb window and domainogram colored by interaction frequency. Right side depicts a model for the toplogy of the locus. ( c ) Same as (b) for GBM3565 ( EGFR .
    CCCTC binding factor CTCF and its paralog the Brother of the Regulator of Imprinted Sites BORIS are highly conserved transcription factors that regulate transcriptional activation and repression insulator function and imprinting control regions ICRs 1 4 Although they have divergent amino and carboxy termini both proteins contain 11 conserved zinc finger domains that work in combination to bind the same DNA elements 1 CTCF is ubiquitously expressed and contributes to transcriptional regulation of cell growth regulated genes including c myc p19 ARF p16 INK4A BRCA1 p53 p27 E2F1 and TERT 1 CTCF also binds to and is required for the enhancer blocking activity of all known insulator elements and ICRs including the H19 IgF2 Prader Willi Angelman syndrome and Inactive X Specific Transcript XIST anti sense loci 5 7 CTCF DNA binding is sensitive to DNA methylation a mark that determines selection of the imprinted allele maternal vs paternal 1 The various functions of CTCF are regulated by at least two different post translational modifications Poly ADP ribosyl ation of CTCF is required for insulator function 8 Phosphorylation of Ser612 by protein kinase CK2 facilitates a switch of CTCF from a transcriptional repressor to an activator at the c myc promoter 9 CTCF mutations or deletions have been found in many breast prostate and Wilms tumors 10 11 Expression of BORIS is restricted to spermatocytes and is mutually exclusive of CTCF 3 In cells expressing BORIS promoters of X linked cancer testis antigens like MAGE 1A are demethylated and activated but methylated and inactive in CTCF expressing somatic cells 12 Like other testis specific proteins BORIS is abnormally expressed in different cancers such as breast cancer and has a greater affinity than CTCF for DNA binding sites detracting from CTCF s potential tumor suppressing activity 1 3 13 14
    https://www.bioz.com/result/anti ctcf/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti ctcf - by Bioz Stars, 2021-07
    94/100 stars

    Images

    1) Product Images from "Functional enhancers shape extrachromosomal oncogene amplifications"

    Article Title: Functional enhancers shape extrachromosomal oncogene amplifications

    Journal: Cell

    doi: 10.1016/j.cell.2019.10.039

    Topology of the EGFR locus in unamplified and amplified glioblastoma. ( a ) (Top) HiC contact map of unamplified glioblastoma model cell line G567. (Middle) CTCF ChlP-seq track of unamplified glioblastoma model cell line GSC23. The red and green arrows denote the orientation of the CTCF motif. (Bottom) Schematic of the boundaries of the loop domain. ( b ) 4C-seq profiles anchored from near the EGFR transcription start site (dotted line) showing interactions to the EGFR enhancers (boxed) for GSC23 (unamplified). Red arcs denote regions with strong distal interactions with EGFR at a 12 kb window thresholded at a 0.12 interaction frequency. H3K27ac ChIP-seq tracks accompany the 4C-seq data. The 4C-seq tracks consist of a main trend of the chromatin interactions based on a 5 kb window and domainogram colored by interaction frequency. Right side depicts a model for the toplogy of the locus. ( c ) Same as (b) for GBM3565 ( EGFR .
    Figure Legend Snippet: Topology of the EGFR locus in unamplified and amplified glioblastoma. ( a ) (Top) HiC contact map of unamplified glioblastoma model cell line G567. (Middle) CTCF ChlP-seq track of unamplified glioblastoma model cell line GSC23. The red and green arrows denote the orientation of the CTCF motif. (Bottom) Schematic of the boundaries of the loop domain. ( b ) 4C-seq profiles anchored from near the EGFR transcription start site (dotted line) showing interactions to the EGFR enhancers (boxed) for GSC23 (unamplified). Red arcs denote regions with strong distal interactions with EGFR at a 12 kb window thresholded at a 0.12 interaction frequency. H3K27ac ChIP-seq tracks accompany the 4C-seq data. The 4C-seq tracks consist of a main trend of the chromatin interactions based on a 5 kb window and domainogram colored by interaction frequency. Right side depicts a model for the toplogy of the locus. ( c ) Same as (b) for GBM3565 ( EGFR .

    Techniques Used: Amplification, Chromatin Immunoprecipitation

    2) Product Images from "The transcription factor musculin promotes the unidirectional development of peripheral Treg cells by suppressing the TH2 transcriptional program"

    Article Title: The transcription factor musculin promotes the unidirectional development of peripheral Treg cells by suppressing the TH2 transcriptional program

    Journal: Nature immunology

    doi: 10.1038/ni.3667

    MSC suppresses T H 2 response within iT regs via regulating T H 2 chromatin conformations (a) Schematic representation of the T H 2 cytokine locus with the positions of indicated genes location conserve non-coding sequences (CNS) and DNase I–hypersensitive sites (RHS and HS). Below, B gl II restriction fragments used in the chromosome conformation capture analysis assay; Relative cross-linking frequencies between a fixed anchor fragment bearing the Il4 gene promoter (upper) or the RHS7 site (lower) and other B gl II fragments via (b) naive T, T H 2, and iT regs , (c) WT and Msc −/− iT regs . The positions of anchor fragments are shown as black shading. The positions of the B gl II fragments cross-linked to the anchor fragments are shown as gray shading; (d) Schematic representation of the T H 2 cytokine locus and CTCF binding sites (CBS-1 to CBS-7); The binding of (e) CTCF or (f) Rad21 to the T H 2 locus on WT and Msc −/− iT regs was determined by ChIP-PCR; Data are pooled from three independent experiments. * P
    Figure Legend Snippet: MSC suppresses T H 2 response within iT regs via regulating T H 2 chromatin conformations (a) Schematic representation of the T H 2 cytokine locus with the positions of indicated genes location conserve non-coding sequences (CNS) and DNase I–hypersensitive sites (RHS and HS). Below, B gl II restriction fragments used in the chromosome conformation capture analysis assay; Relative cross-linking frequencies between a fixed anchor fragment bearing the Il4 gene promoter (upper) or the RHS7 site (lower) and other B gl II fragments via (b) naive T, T H 2, and iT regs , (c) WT and Msc −/− iT regs . The positions of anchor fragments are shown as black shading. The positions of the B gl II fragments cross-linked to the anchor fragments are shown as gray shading; (d) Schematic representation of the T H 2 cytokine locus and CTCF binding sites (CBS-1 to CBS-7); The binding of (e) CTCF or (f) Rad21 to the T H 2 locus on WT and Msc −/− iT regs was determined by ChIP-PCR; Data are pooled from three independent experiments. * P

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction

    3) Product Images from "Role of CTCF in Regulating SLC45A3-ELK4 Chimeric RNA"

    Article Title: Role of CTCF in Regulating SLC45A3-ELK4 Chimeric RNA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0150382

    AR and CTCF do not colocalize, or interact in LNCaP cells. (A) Re-ChIP by CTCF, AR antibodies, or control IgG. Insulator1, 2 and, ARE1, 2, and 3 were tested. (B) Co-immunoprecipitation by CTCF antibody, and western blotting by AR, CTCF, or GAPDH. R-RWPE-1, L-LNCaP, R1881-synthetic androgen.
    Figure Legend Snippet: AR and CTCF do not colocalize, or interact in LNCaP cells. (A) Re-ChIP by CTCF, AR antibodies, or control IgG. Insulator1, 2 and, ARE1, 2, and 3 were tested. (B) Co-immunoprecipitation by CTCF antibody, and western blotting by AR, CTCF, or GAPDH. R-RWPE-1, L-LNCaP, R1881-synthetic androgen.

    Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation, Western Blot

    4) Product Images from "CTCF Governs the Identity and Migration of MGE-Derived Cortical Interneurons"

    Article Title: CTCF Governs the Identity and Migration of MGE-Derived Cortical Interneurons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3496-17.2018

    CTCF functions independently of Nkx2.1 to regulate Lhx6 and Lhx8 . A , Western blot of Nkx2.1 in E13.5 telencephalon of controls and Ctcf Nes-Cre (KO) embryos. β-actin was used for normalization. Quantification is shown graphically on the right. Error bars indicate SEM. p = 0.968, Student's t test. B , Nkx2.1 immunofluorescence staining (red) in E13.5 coronal sections. Nuclei were counterstained with DAPI (blue). Scale bar, 100 μm. C , Co-IP of CTCF and Nkx2.1 of pooled wild-type MGE tissue. Asterisks indicate nonspecific IgG band. D , Venn diagram showing the overlap of CTCF and Nkx2.1 ChIP-Seq peaks in the MGE ( p
    Figure Legend Snippet: CTCF functions independently of Nkx2.1 to regulate Lhx6 and Lhx8 . A , Western blot of Nkx2.1 in E13.5 telencephalon of controls and Ctcf Nes-Cre (KO) embryos. β-actin was used for normalization. Quantification is shown graphically on the right. Error bars indicate SEM. p = 0.968, Student's t test. B , Nkx2.1 immunofluorescence staining (red) in E13.5 coronal sections. Nuclei were counterstained with DAPI (blue). Scale bar, 100 μm. C , Co-IP of CTCF and Nkx2.1 of pooled wild-type MGE tissue. Asterisks indicate nonspecific IgG band. D , Venn diagram showing the overlap of CTCF and Nkx2.1 ChIP-Seq peaks in the MGE ( p

    Techniques Used: Western Blot, Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Chromatin Immunoprecipitation

    5) Product Images from "Analysis of neonatal brain lacking ATRX or MeCP2 reveals changes in nucleosome density, CTCF binding and chromatin looping"

    Article Title: Analysis of neonatal brain lacking ATRX or MeCP2 reveals changes in nucleosome density, CTCF binding and chromatin looping

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku564

    MeCP2 is required for ATRX and CTCF binding to the H19 ICR and the long-range chromatin interactions across the H19/Igf2 domain. ( a ) ATRX ChIP was performed in control and MeCP2 null neonatal forebrain and shows that MeCP2 is required for ATRX occupancy at the H19 ICR. The graph shows mean fold change value ( n = 4) and error bars depict SEM. ( b ) Diagram of the H19 ICR and location of the H19-1 and H19-3 primer pairs used in the ChIP-qPCR in (a). ( c ) Allelic nucleosome digestion assay of the H19 ICR was performed in control and MeCP2 null forebrains and reveals increased protection at the 5′ end of the maternal H19 ICR in the MeCP2 null neonatal forebrain. A significant increase in nucleosome occupancy was observed within regions B and I ( P = 0.042) of the H19 ICR and a significant decrease at site E ( P = 0.05). Graphs depict mean fold change and statistical analysis was performed by a two-tailed t -test ( n = 3, errors bars depict SEM). * P
    Figure Legend Snippet: MeCP2 is required for ATRX and CTCF binding to the H19 ICR and the long-range chromatin interactions across the H19/Igf2 domain. ( a ) ATRX ChIP was performed in control and MeCP2 null neonatal forebrain and shows that MeCP2 is required for ATRX occupancy at the H19 ICR. The graph shows mean fold change value ( n = 4) and error bars depict SEM. ( b ) Diagram of the H19 ICR and location of the H19-1 and H19-3 primer pairs used in the ChIP-qPCR in (a). ( c ) Allelic nucleosome digestion assay of the H19 ICR was performed in control and MeCP2 null forebrains and reveals increased protection at the 5′ end of the maternal H19 ICR in the MeCP2 null neonatal forebrain. A significant increase in nucleosome occupancy was observed within regions B and I ( P = 0.042) of the H19 ICR and a significant decrease at site E ( P = 0.05). Graphs depict mean fold change and statistical analysis was performed by a two-tailed t -test ( n = 3, errors bars depict SEM). * P

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Two Tailed Test

    ATRX regulates nucleosome occupancy and CTCF binding at the H19 ICR. ( a ) Schematic representation of the H19 ICR and alignment of primers used for ChIP-qPCR (top) and nucleosome occupancy analysis (bottom). Gray boxes indicate the positions of CTCF binding sites and a red box marks the ATRX-binding site. Numbers indicate the relative position from the start of the H19 ICR. ( b ) ChIP of ATRX in E13.5 and P0.5 forebrains shows binding of ATRX in the middle portion of the H19 ICR (H19-3) at P0.5, as observed previously, but not at E13.5, indicating that ATRX is recruited to the H19 ICR in the late embryonic/neonatal brain. ( c ) CTCF ChIP at E13.5 and P0.5 shows binding of CTCF at both time points at previously identified areas of occupancy (H19-2 and H19-4). ( d ) ChIP of CTCF in control and ATRX-null forebrain tissue at E13.5 and P0.5 reveals that ATRX binding is required for CTCF binding at P0.5, but not at E13.5. Graphs in (b), (c) and (d) represent mean values, n = 3 for each and error bars depict SEM. ( e ) Diagram depicting the methodology used for allele-specific micrococcal nuclease digestion. Empty circles indicate unmethylated CpGs and black circles indicate methylated CpGs. ( f ) Validation of allele-specificity of nucleosome occupancy protocol, in which the methylated paternal H19 ICR sequence is digested by McrBC. In F1 polymorphic 129Sv (maternal)/castaneous (paternal) forebrain samples, MfeI digests 129Sv maternal DNA and McrBC digests methylated paternal DNA. Following digestion, DNA was amplified using primers spanning the MfeI restriction site. ( g ) qPCR of micrococcal nuclease and McrBC-digested DNA reveals increased protection at the 5′ end of the maternal H19 ICR in the ATRX-null samples. A significant increase in nucleosome occupancy was observed at regions B ( P = 0.016) and C ( P = 0.05) of the H19 ICR and a significant decrease at adjacent site G ( P = 0.001). Graph shows mean fold change and statistical analysis was performed by a two-tailed t -test ( n = 3, errors bars depict SEM). * P
    Figure Legend Snippet: ATRX regulates nucleosome occupancy and CTCF binding at the H19 ICR. ( a ) Schematic representation of the H19 ICR and alignment of primers used for ChIP-qPCR (top) and nucleosome occupancy analysis (bottom). Gray boxes indicate the positions of CTCF binding sites and a red box marks the ATRX-binding site. Numbers indicate the relative position from the start of the H19 ICR. ( b ) ChIP of ATRX in E13.5 and P0.5 forebrains shows binding of ATRX in the middle portion of the H19 ICR (H19-3) at P0.5, as observed previously, but not at E13.5, indicating that ATRX is recruited to the H19 ICR in the late embryonic/neonatal brain. ( c ) CTCF ChIP at E13.5 and P0.5 shows binding of CTCF at both time points at previously identified areas of occupancy (H19-2 and H19-4). ( d ) ChIP of CTCF in control and ATRX-null forebrain tissue at E13.5 and P0.5 reveals that ATRX binding is required for CTCF binding at P0.5, but not at E13.5. Graphs in (b), (c) and (d) represent mean values, n = 3 for each and error bars depict SEM. ( e ) Diagram depicting the methodology used for allele-specific micrococcal nuclease digestion. Empty circles indicate unmethylated CpGs and black circles indicate methylated CpGs. ( f ) Validation of allele-specificity of nucleosome occupancy protocol, in which the methylated paternal H19 ICR sequence is digested by McrBC. In F1 polymorphic 129Sv (maternal)/castaneous (paternal) forebrain samples, MfeI digests 129Sv maternal DNA and McrBC digests methylated paternal DNA. Following digestion, DNA was amplified using primers spanning the MfeI restriction site. ( g ) qPCR of micrococcal nuclease and McrBC-digested DNA reveals increased protection at the 5′ end of the maternal H19 ICR in the ATRX-null samples. A significant increase in nucleosome occupancy was observed at regions B ( P = 0.016) and C ( P = 0.05) of the H19 ICR and a significant decrease at adjacent site G ( P = 0.001). Graph shows mean fold change and statistical analysis was performed by a two-tailed t -test ( n = 3, errors bars depict SEM). * P

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Methylation, Sequencing, Amplification, Two Tailed Test

    Model of ATRX and MeCP2 function. ( a ) In the wild-type brain, MeCP2 recruits ATRX to the maternal H19 ICR in the late embryonic/neonatal period. ATRX translocates along the chromatin fiber and alters nucleosome positioning to generate an extended linker region and promote CTCF occupancy. CTCF then dictates intra chromosomal interactions. ( b ) In the absence of ATRX, increased nucleosome occupancy disrupts CTCF binding, leading to a loss of intra chromosomal interactions.
    Figure Legend Snippet: Model of ATRX and MeCP2 function. ( a ) In the wild-type brain, MeCP2 recruits ATRX to the maternal H19 ICR in the late embryonic/neonatal period. ATRX translocates along the chromatin fiber and alters nucleosome positioning to generate an extended linker region and promote CTCF occupancy. CTCF then dictates intra chromosomal interactions. ( b ) In the absence of ATRX, increased nucleosome occupancy disrupts CTCF binding, leading to a loss of intra chromosomal interactions.

    Techniques Used: Binding Assay

    6) Product Images from "Identification of a Novel Enhancer/Chromatin Opening Element Associated with High-Level γ-Globin Gene Expression"

    Article Title: Identification of a Novel Enhancer/Chromatin Opening Element Associated with High-Level γ-Globin Gene Expression

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00197-18

    Reduced γ-globin expression in K562 cells exposed to the HBG-4kb ZF. (A) Temporal effect of the HBG-4kb ZF on gene expression in K562 cells. K562 cells were exposed to 500 nM HBG-4kb ZF for 6 to 24 h, as indicated. Untreated cells were also examined as indicated. Cells were lysed, the lysates were compartmentalized into nuclear and cytoplasmic fractions, and the proteins were subjected to Western blotting using antibodies specific for CTCF, GAPDH, HBG-4kb ZF (ZF-DBD), γ-globin, Brg1, and GATA1, as shown. (B) Effect of the HBG-4kb ZF on γ-globin and GATA1 RNA and protein levels during the course of exposure to the HBG-4kb ZF. Proteins and RNA were extracted and subjected to Western blotting (using γ-globin- and GATA1-specific antibodies) or RT-qPCR using primers specific for the γ-globin (HBG) or GATA1 genes. The RT-qPCR data were from three independent experiments, and the error bars represent the SEM. (C) Assessment of ZF-DBD dosage effect. K562 cells were exposed to different concentrations (5, 50, and 500 nM) of the HBG-4kb ZF for 24 or 48 h. After exposure to the ZF-DBD, the K562 cells were subjected to Western blotting experiments with antibodies specific for GAPDH, γ-globin, or the HBG-4kb ZF (αZF). The graph on the bottom reflects the quantitation of signals from 3 independent experiments, with the error bars representing the SEM. (D) Exposure of K562 cells to the HBG-4kb ZF specifically reduced γ-globin gene expression. K562 cells were exposed to 500 nM HBG-4kb ZF (top) or to 500 nM negative-control ZF-DBD (bottom) for 12 h. RNA was isolated, reverse transcribed, and subjected to qPCR using primers specific for the γ-globin (HBGs), Gγ-globin (HBG2), adult β-globin (HBB), embryonic ε-globin (HBE), and GATA1 genes, as indicated. Expression in K562 cells not exposed to the ZF-DBDs (Untreated) was set at 1. Error bars reflect the SEMs from three independent biological repeats (**, P
    Figure Legend Snippet: Reduced γ-globin expression in K562 cells exposed to the HBG-4kb ZF. (A) Temporal effect of the HBG-4kb ZF on gene expression in K562 cells. K562 cells were exposed to 500 nM HBG-4kb ZF for 6 to 24 h, as indicated. Untreated cells were also examined as indicated. Cells were lysed, the lysates were compartmentalized into nuclear and cytoplasmic fractions, and the proteins were subjected to Western blotting using antibodies specific for CTCF, GAPDH, HBG-4kb ZF (ZF-DBD), γ-globin, Brg1, and GATA1, as shown. (B) Effect of the HBG-4kb ZF on γ-globin and GATA1 RNA and protein levels during the course of exposure to the HBG-4kb ZF. Proteins and RNA were extracted and subjected to Western blotting (using γ-globin- and GATA1-specific antibodies) or RT-qPCR using primers specific for the γ-globin (HBG) or GATA1 genes. The RT-qPCR data were from three independent experiments, and the error bars represent the SEM. (C) Assessment of ZF-DBD dosage effect. K562 cells were exposed to different concentrations (5, 50, and 500 nM) of the HBG-4kb ZF for 24 or 48 h. After exposure to the ZF-DBD, the K562 cells were subjected to Western blotting experiments with antibodies specific for GAPDH, γ-globin, or the HBG-4kb ZF (αZF). The graph on the bottom reflects the quantitation of signals from 3 independent experiments, with the error bars representing the SEM. (D) Exposure of K562 cells to the HBG-4kb ZF specifically reduced γ-globin gene expression. K562 cells were exposed to 500 nM HBG-4kb ZF (top) or to 500 nM negative-control ZF-DBD (bottom) for 12 h. RNA was isolated, reverse transcribed, and subjected to qPCR using primers specific for the γ-globin (HBGs), Gγ-globin (HBG2), adult β-globin (HBB), embryonic ε-globin (HBE), and GATA1 genes, as indicated. Expression in K562 cells not exposed to the ZF-DBDs (Untreated) was set at 1. Error bars reflect the SEMs from three independent biological repeats (**, P

    Techniques Used: Expressing, Western Blot, Quantitative RT-PCR, Quantitation Assay, Negative Control, Isolation, Real-time Polymerase Chain Reaction

    7) Product Images from "Mapping Human Transient Transcriptomes Using Single Nucleotide Resolution 4sU Sequencing (SNU-Seq)"

    Article Title: Mapping Human Transient Transcriptomes Using Single Nucleotide Resolution 4sU Sequencing (SNU-Seq)

    Journal: bioRxiv

    doi: 10.1101/2021.07.14.452379

    SNU-seq identifies IFNγ-induced changes in the enhancer landscape. A , C Venn diagrams showing IFNγ-induced transcriptional changes within a 5kb window surrounding FANTOM5 enhancers ( A ) or ATAC-seq peaks ( C ) for each condition; untreated vs 0.5 hrs (pink), 2 hrs (blue) or 24 hrs (green) of IFNγ treatment. B , D IGV images of the regions surround the IRF1-AS1 locus ( B ) or a 12kb stretch of chr3 with no annotated loci ( D ) showing strand-specific SNU-Seq, RNA-Seq, DNA accessibility (ATAC-Seq) and ChIP-Seq profiles of CTCF, H3K27ac and H3K4me3 data for each treatment sample (n=2-3), with the untreated sample in black, and 0.5, 2 and 24 hrs of IFNγ treatment in pink, blue and green, respectively. All tracks are spike-in normalised except ATAC-Seq which is normalised based on sequencing depth. Annotated FANTOM5 enhancers are represented by the blue boxes.
    Figure Legend Snippet: SNU-seq identifies IFNγ-induced changes in the enhancer landscape. A , C Venn diagrams showing IFNγ-induced transcriptional changes within a 5kb window surrounding FANTOM5 enhancers ( A ) or ATAC-seq peaks ( C ) for each condition; untreated vs 0.5 hrs (pink), 2 hrs (blue) or 24 hrs (green) of IFNγ treatment. B , D IGV images of the regions surround the IRF1-AS1 locus ( B ) or a 12kb stretch of chr3 with no annotated loci ( D ) showing strand-specific SNU-Seq, RNA-Seq, DNA accessibility (ATAC-Seq) and ChIP-Seq profiles of CTCF, H3K27ac and H3K4me3 data for each treatment sample (n=2-3), with the untreated sample in black, and 0.5, 2 and 24 hrs of IFNγ treatment in pink, blue and green, respectively. All tracks are spike-in normalised except ATAC-Seq which is normalised based on sequencing depth. Annotated FANTOM5 enhancers are represented by the blue boxes.

    Techniques Used: RNA Sequencing Assay, Chromatin Immunoprecipitation, Sequencing

    8) Product Images from "Characterization of Human Telomerase Reverse Transcriptase Promoter Methylation and Transcription Factor Binding in Differentiated Thyroid Cancer Cell Lines"

    Article Title: Characterization of Human Telomerase Reverse Transcriptase Promoter Methylation and Transcription Factor Binding in Differentiated Thyroid Cancer Cell Lines

    Journal: Genes, chromosomes & cancer

    doi: 10.1002/gcc.22735

    TERT promoter methylation. Methylation pattern relative to the transcription start site (TSS) in either cell lines (CL) or patient blood/tumor samples (PS) with positive telomerase expression given in black and white rectangles, with black denoting hypermethylated regions and white denoting hypomethylated regions. Cancer abbreviations are: breast (BRCA), lung (LUN), colon (COL), cervical (CES), acute myeloid leukemia (AML), hepatocellular carcinoma (HCC), acute promyelocytic leukemia (APL), bladder (BLA), and glioblastoma (GBM). Transcription factor binding sites for MYC, ETS, and CTCF are shown, with the minimal promoter represented by a dashed arrow, and TERT promoter mutations indicated by *. The proximal MYC site is shaded to denote a lesser importance in transcriptional activation.1 The translation start site is depicted by a dashed arrow head. The amplicons designed to tile the promoter are shown by the dark double arrowhead.
    Figure Legend Snippet: TERT promoter methylation. Methylation pattern relative to the transcription start site (TSS) in either cell lines (CL) or patient blood/tumor samples (PS) with positive telomerase expression given in black and white rectangles, with black denoting hypermethylated regions and white denoting hypomethylated regions. Cancer abbreviations are: breast (BRCA), lung (LUN), colon (COL), cervical (CES), acute myeloid leukemia (AML), hepatocellular carcinoma (HCC), acute promyelocytic leukemia (APL), bladder (BLA), and glioblastoma (GBM). Transcription factor binding sites for MYC, ETS, and CTCF are shown, with the minimal promoter represented by a dashed arrow, and TERT promoter mutations indicated by *. The proximal MYC site is shaded to denote a lesser importance in transcriptional activation.1 The translation start site is depicted by a dashed arrow head. The amplicons designed to tile the promoter are shown by the dark double arrowhead.

    Techniques Used: Methylation, Expressing, Binding Assay, Activation Assay

    Transcription factor binding varies at the TERT promoter in DTC cell lines and normal thyroid tissue. Binding was measured relative to 5% input chromatin, and binding at the TERT locus normalized to a positive control locus for each factor in the indicated cell lines and normal thyroid tissue by ChIP-qPCR for (A) CTCF, (B) GABPA, (C) MYC, at two positions- the minimal promoter binding site and the upstream binding site, (D) GSC. All error bars represent standard error within triplicates.
    Figure Legend Snippet: Transcription factor binding varies at the TERT promoter in DTC cell lines and normal thyroid tissue. Binding was measured relative to 5% input chromatin, and binding at the TERT locus normalized to a positive control locus for each factor in the indicated cell lines and normal thyroid tissue by ChIP-qPCR for (A) CTCF, (B) GABPA, (C) MYC, at two positions- the minimal promoter binding site and the upstream binding site, (D) GSC. All error bars represent standard error within triplicates.

    Techniques Used: Binding Assay, Positive Control, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    9) Product Images from "Induction of the Cellular MicroRNA, Hs_154, by West Nile Virus Contributes to Virus-Mediated Apoptosis through Repression of Antiapoptotic Factors"

    Article Title: Induction of the Cellular MicroRNA, Hs_154, by West Nile Virus Contributes to Virus-Mediated Apoptosis through Repression of Antiapoptotic Factors

    Journal: Journal of Virology

    doi: 10.1128/JVI.06883-11

    Overexpression of CTCF or ECOP or a Hs_154-targeting LNA protects cells from WNV-mediated apoptosis. (A) HEK293 cells were transfected with empty vector (control) or plasmids encoding CTCF or FLAG-tagged ECOP. At 48 h posttransfection, cells were WNV
    Figure Legend Snippet: Overexpression of CTCF or ECOP or a Hs_154-targeting LNA protects cells from WNV-mediated apoptosis. (A) HEK293 cells were transfected with empty vector (control) or plasmids encoding CTCF or FLAG-tagged ECOP. At 48 h posttransfection, cells were WNV

    Techniques Used: Over Expression, Transfection, Plasmid Preparation

    10) Product Images from "Transcription-driven cohesin repositioning rewires chromatin loops in cellular senescence"

    Article Title: Transcription-driven cohesin repositioning rewires chromatin loops in cellular senescence

    Journal: bioRxiv

    doi: 10.1101/823831

    Correlation between cohesin redistribution and loop rewiring during RIS: a , Number of CTCF and cohesin ChIP-seq peaks with increased (green) and decreased (blue) binding. b , Position of CTCF and cohesin binding changes relative to the growing IMR90 loops 6 ; each loop is represented as a radial segment linking the two loop anchors. c , Difference between RIS and growing aggregated Hi-C interactions neighbourhoods (20 kb resolution) of IMR90 loops overlapping with significantly decreased cohesin binding at least one loop ends (left). Compare to all IMR90 loops with significantly decreased interactions during RIS (right). d , De novo cohesin (RAD21 and SMC3) binding at the 3’ end of IL1B coinciding with the inferred position of the de novo loop formation in RIS, as well as CTCF and H3K27ac THOR-normalized ChIP-seq signal in growing (Grow) and RIS. e , THOR-normalized SMC3 ChIP-seq signal at the IL1 locus in RIS via constitutive expression of HRAS G12V (pBabe) in IMR90 and WI38 cells, as well as matched growing controls (Vector). f , THOR-normalized ChIP-seq signal of cohesin (RAD21 and SMC3) and CTCF at the IL1 locus in TNFα-treated and matched control IMR90 cells. g , Proposed model for the de novo loop formation at the IL1 locus, separating IL1B from IL1A and CKAP2L , along with their specific enhancers.
    Figure Legend Snippet: Correlation between cohesin redistribution and loop rewiring during RIS: a , Number of CTCF and cohesin ChIP-seq peaks with increased (green) and decreased (blue) binding. b , Position of CTCF and cohesin binding changes relative to the growing IMR90 loops 6 ; each loop is represented as a radial segment linking the two loop anchors. c , Difference between RIS and growing aggregated Hi-C interactions neighbourhoods (20 kb resolution) of IMR90 loops overlapping with significantly decreased cohesin binding at least one loop ends (left). Compare to all IMR90 loops with significantly decreased interactions during RIS (right). d , De novo cohesin (RAD21 and SMC3) binding at the 3’ end of IL1B coinciding with the inferred position of the de novo loop formation in RIS, as well as CTCF and H3K27ac THOR-normalized ChIP-seq signal in growing (Grow) and RIS. e , THOR-normalized SMC3 ChIP-seq signal at the IL1 locus in RIS via constitutive expression of HRAS G12V (pBabe) in IMR90 and WI38 cells, as well as matched growing controls (Vector). f , THOR-normalized ChIP-seq signal of cohesin (RAD21 and SMC3) and CTCF at the IL1 locus in TNFα-treated and matched control IMR90 cells. g , Proposed model for the de novo loop formation at the IL1 locus, separating IL1B from IL1A and CKAP2L , along with their specific enhancers.

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Hi-C, Expressing, Plasmid Preparation

    11) Product Images from "NF-?B Subtypes Regulate CCCTC Binding Factor Affecting Corneal Epithelial Cell Fate *"

    Article Title: NF-?B Subtypes Regulate CCCTC Binding Factor Affecting Corneal Epithelial Cell Fate *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.094425

    Effects of knocking down NF-κB p50 and p65 on UV stress-induced CTCF suppression and apoptosis. Knockdown of NF-κB p50 with specific siRNA markedly affected UV stress-induced CTCF suppression ( A ), caspase 3 activation ( B ), and cell death
    Figure Legend Snippet: Effects of knocking down NF-κB p50 and p65 on UV stress-induced CTCF suppression and apoptosis. Knockdown of NF-κB p50 with specific siRNA markedly affected UV stress-induced CTCF suppression ( A ), caspase 3 activation ( B ), and cell death

    Techniques Used: Activation Assay

    Related Articles

    Binding Assay:

    Article Title: CTCF-Mediated and Pax6-Associated Gene Expression in Corneal Epithelial Cell-Specific Differentiation
    Article Snippet: These results indicate that CTCF-mediated chromatin remodeling of PAX6 and PAX6 -associated genes, such as RCN1 and ADAM17 , are indeed occurred in the process of HTCE cell differentiation. .. Effect of altered CTCF binding activity on PAX6 and PAX6 -associated gene interactions To verify the specific effect of differentiation on CTCF binding capability in promoter regions of PAX6 , RCN1 and ADAM17 in HTCE cells, we performed chromatin immunoprecipitation (ChIP) using specific antibodies against CTCF (Cell signaling). .. As it has been described in details of ChIP protocol in Methods, several pairs of the primers were used in ChIP-based PCRs for PAX6 , RCN1 and ADAM17 to generate 250 to 300 bp DNA fragments, respectively.

    Activity Assay:

    Article Title: CTCF-Mediated and Pax6-Associated Gene Expression in Corneal Epithelial Cell-Specific Differentiation
    Article Snippet: These results indicate that CTCF-mediated chromatin remodeling of PAX6 and PAX6 -associated genes, such as RCN1 and ADAM17 , are indeed occurred in the process of HTCE cell differentiation. .. Effect of altered CTCF binding activity on PAX6 and PAX6 -associated gene interactions To verify the specific effect of differentiation on CTCF binding capability in promoter regions of PAX6 , RCN1 and ADAM17 in HTCE cells, we performed chromatin immunoprecipitation (ChIP) using specific antibodies against CTCF (Cell signaling). .. As it has been described in details of ChIP protocol in Methods, several pairs of the primers were used in ChIP-based PCRs for PAX6 , RCN1 and ADAM17 to generate 250 to 300 bp DNA fragments, respectively.

    Chromatin Immunoprecipitation:

    Article Title: CTCF-Mediated and Pax6-Associated Gene Expression in Corneal Epithelial Cell-Specific Differentiation
    Article Snippet: These results indicate that CTCF-mediated chromatin remodeling of PAX6 and PAX6 -associated genes, such as RCN1 and ADAM17 , are indeed occurred in the process of HTCE cell differentiation. .. Effect of altered CTCF binding activity on PAX6 and PAX6 -associated gene interactions To verify the specific effect of differentiation on CTCF binding capability in promoter regions of PAX6 , RCN1 and ADAM17 in HTCE cells, we performed chromatin immunoprecipitation (ChIP) using specific antibodies against CTCF (Cell signaling). .. As it has been described in details of ChIP protocol in Methods, several pairs of the primers were used in ChIP-based PCRs for PAX6 , RCN1 and ADAM17 to generate 250 to 300 bp DNA fragments, respectively.

    Article Title: Contribution of CTCF binding to transcriptional activity at the HOXA locus in NPM1-mutant AML cells
    Article Snippet: Kasumi-1, IMS-MS2, and MOLM13 cell lines were cultured in RPMI-1640 with 1% penicillin-streptomycin and fetal calf serum (20% for Kasumi-1 and MOLM13, 10% for IMS-MS2). .. ChIP-seq preparation and analysisChIP-seq was performed using the ChIPmentation method ( ) with the following ChIP-grade antibodies: CTCF (2899S), H3K27me3 (9733S), and H3K27ac (8173S) from Cell Signaling Technology and H3K4me3 (ab1012) from Abcam. .. All libraries were sequenced on a NovaSeq 6000 (Illumina, San Diego, CA) to obtain ∼50 million 150 bp paired end reads, and data were analyzed via adapter trimming with trim galore and alignment to the GRCh38 human reference sequence using bwa mem ( ).

    Article Title: Methods for Scarless, Selection-Free Generation of Human Cells and Allele-Specific Functional Analysis of Disease-Associated SNPs and Variants of Uncertain Significance
    Article Snippet: DNA was sheared via sonication using the Bioruptor 2000 (Diagenode). .. For each ChIP, 3 volumes of IP Dilution Buffer (16.7 mM Tris-HCl, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton × 100, 0.01% SDS, 1x Protease Inhibitors; pH 8.0) was added to 20ug of chromatin and 2ug of antibody (TCF7L2 (D31H2); CTCF (C48H11), Cell Signaling Technology, Inc.). ..

    Article Title: The transcription factor musculin promotes the unidirectional development of peripheral Treg cells by suppressing the TH2 transcriptional program
    Article Snippet: ChIP was undertaken following manufacturer’s instructions (Cell signaling) followed by PCR amplification of the immunoprecipitates in defined genomic regions. .. The following antibodies were used for ChIP: anti-Smad3 (ab28379, Abcam), anti-GATA3 (D-16, Santa Cruz), anti-MSC (F-20, Santa Cruz), anti-CTCF (D31H2, Cell signaling), anti-Rad21 (ab992, Abcam), anti-acetylated H4 (06–866, Millipore), anti-H3K9me3 (C5B11, Cell Signaling) and anti-H3K27me3 (07–449, Millipore). .. The regions that were identified by ChIP PCR were amplified by predefined primers for the genes including CBS, Foxp3, Il-4, Il-5, Il-13 and Msc .

    Lysis:

    Article Title: Genomic Space of MGMT in Human Glioma Revisited: Novel Motifs, Regulatory RNAs, NRF1, 2, and CTCF Involvement in Gene Expression
    Article Snippet: Chromatin Immunoprecipitation (ChIP) Assay The EpiQuik ChIP assay kit (Epigentek, Farmingdale, NY, USA) was used to determine the presence of a CTCF-binding site in the MGMT-E1 promoter. .. Briefly, the SF-188 GBM cells were exposed to 1% formaldehyde in the medium without serum for 10 min, followed by lysis, DNA shearing, and protein/DNA Immunoprecipitation using CTCF antibodies (Cell Signaling Technology, Danvers, MA, USA). .. The antibodies to RNA polymerase were used as a positive control and the normal mouse IgG as a negative control.

    Immunoprecipitation:

    Article Title: Genomic Space of MGMT in Human Glioma Revisited: Novel Motifs, Regulatory RNAs, NRF1, 2, and CTCF Involvement in Gene Expression
    Article Snippet: Chromatin Immunoprecipitation (ChIP) Assay The EpiQuik ChIP assay kit (Epigentek, Farmingdale, NY, USA) was used to determine the presence of a CTCF-binding site in the MGMT-E1 promoter. .. Briefly, the SF-188 GBM cells were exposed to 1% formaldehyde in the medium without serum for 10 min, followed by lysis, DNA shearing, and protein/DNA Immunoprecipitation using CTCF antibodies (Cell Signaling Technology, Danvers, MA, USA). .. The antibodies to RNA polymerase were used as a positive control and the normal mouse IgG as a negative control.

    ChIP-sequencing:

    Article Title: Contribution of CTCF binding to transcriptional activity at the HOXA locus in NPM1-mutant AML cells
    Article Snippet: Kasumi-1, IMS-MS2, and MOLM13 cell lines were cultured in RPMI-1640 with 1% penicillin-streptomycin and fetal calf serum (20% for Kasumi-1 and MOLM13, 10% for IMS-MS2). .. ChIP-seq preparation and analysisChIP-seq was performed using the ChIPmentation method ( ) with the following ChIP-grade antibodies: CTCF (2899S), H3K27me3 (9733S), and H3K27ac (8173S) from Cell Signaling Technology and H3K4me3 (ab1012) from Abcam. .. All libraries were sequenced on a NovaSeq 6000 (Illumina, San Diego, CA) to obtain ∼50 million 150 bp paired end reads, and data were analyzed via adapter trimming with trim galore and alignment to the GRCh38 human reference sequence using bwa mem ( ).

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    Cell Signaling Technology Inc anti ctcf rabbit monoclonal antibody
    Transcriptional profiling of genes altered with <t>CTCF</t> knockdown. a Heat map of differentially expressed (DE) transcripts following 5 days of CTCF <t>shRNA</t> induction (Dox) versus uninduced vehicle control (vehicle). CTCF knockdown in HPECE6/E7 leads to 1308 significantly altered gene transcripts (FDR
    Anti Ctcf Rabbit Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Foxg1Cre -mediated deletion of <t>Ctcf</t> results in a massive increase in apoptosis. A , Table of genotypes obtained during the embryonic and postnatal periods. Ratios at each time point were analyzed using a χ 2 test. Het, Ctcf flox/WT ;Foxg1-cre +/− . B , Dark-field images of control and Ctcf Foxg1-cre embryos at E13.5. (Please note limbs were taken for genotyping). C , H E staining of E13.5 sagittal cryosections demonstrates complete loss of cortex (Ctx), hippocampal hem (H), basal ganglia (BG), lens (L), and anterior retina (AR), but not the posterior retina (PR), in Ctcf Foxg1-cre embryos. D , Dark-field images of control and Ctcf Foxg1-cre embryos at E11.5. The dashed circle outlines the telencephalon, which is visibly reduced in size in the Ctcf Foxg1-cre embryos compared to littermate controls. E , Immunodetection of CTCF (red) in E11.5 sagittal cryosections confirms specific loss of CTCF expression in the forebrain neuroepithelium of Ctcf Foxg1-cre embryos. F , Pregnant females were subjected to a 1 h BrdU pulse before being killed. Immunodetection of BrdU in E11.5 control and Ctcf Foxg1-cre cortical neuroepithelium is shown. G , BrdU + cells were counted and expressed as a percentage of the total number of DAPI + cells ( n = 3). H , Immunodetection of activated <t>caspase-3</t> (red) in control and Ctcf Foxg1-cre cortical neuroepithelium at E11.5. I , AC3 + cells were counted and expressed as a percentage of the total number of DAPI + cells ( n = 3). J , TUNEL (green) detection in E11.5 control and Ctcf Foxg1-cre cortical neuroepithelium. Error bars represent the SEM. Original magnification: C , 25×; E , 50×; F , H , J , 200×. Scale bars: C , top, 1 mm; bottom, 400 μm; E , 200 μm; F , H , 50 μm; J , 100 μm.
    Rabbit Anti Ctcf, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc ctcf antibody
    Analysis of the rs4919742-associated chromatin loops. Guide RNAs targeting regions encompassing <t>CTCF</t> site 4 (the PCa risk-associated CTCF site), CTCF site 5, and/or CTCF site 6 (or the empty guide RNA vector as a control) were introduced into <t>22Rv1</t> prostate cancer cells, along with Cas9. Cell pools were harvested, and KRT78 expression was analyzed by RT-qPCR. Shown within the blue bars is the fold change in KRT78 expression in the pools that received guide RNAs vs the vector control. The yellow X indicates which CTCF site has been deleted; the size of each deletion can be found in Additional file 5 : Table S4
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    Transcriptional profiling of genes altered with CTCF knockdown. a Heat map of differentially expressed (DE) transcripts following 5 days of CTCF shRNA induction (Dox) versus uninduced vehicle control (vehicle). CTCF knockdown in HPECE6/E7 leads to 1308 significantly altered gene transcripts (FDR

    Journal: Clinical Epigenetics

    Article Title: CTCF loss mediates unique DNA hypermethylation landscapes in human cancers

    doi: 10.1186/s13148-020-00869-7

    Figure Lengend Snippet: Transcriptional profiling of genes altered with CTCF knockdown. a Heat map of differentially expressed (DE) transcripts following 5 days of CTCF shRNA induction (Dox) versus uninduced vehicle control (vehicle). CTCF knockdown in HPECE6/E7 leads to 1308 significantly altered gene transcripts (FDR

    Article Snippet: CTCF shRNA and controls were analyzed by western blotting using anti-CTCF rabbit monoclonal antibody (Cell Signaling #3418) to verify knockdown.

    Techniques: shRNA

    Knockdown of CTCF protein results in DNA hypermethylation preferentially at CTCF sites. a Workflow of methylated DNA immunoprecipitation followed by copy number array application (MeDIP-chip) for detecting methylation alterations. NspI restriction fragments were bound to anti-5-methylcytosine antibody, eluted, and hybridized to a Affymetrix Cytoscan HD probe. An unenriched total input fraction was processed for comparison. b Short hairpin mediated CTCF knockdown in two separate shRNA targeting CTCF verified by western blotting after 3 and 5 days of shRNA induction including shRNA non-silencing control (shNSC). Data shown are one representative of 3 independent experiments using immortalized HPECs. Percentage knockdown compared to shCTCF -Dox control, quantified by ImageJ. c Volcano plot of detected methylation changes in CTCF knockdown HPECE6/E7 after 5 days of dox exposure (cut-point, methylation Abs. Log2FC > 1.5, P

    Journal: Clinical Epigenetics

    Article Title: CTCF loss mediates unique DNA hypermethylation landscapes in human cancers

    doi: 10.1186/s13148-020-00869-7

    Figure Lengend Snippet: Knockdown of CTCF protein results in DNA hypermethylation preferentially at CTCF sites. a Workflow of methylated DNA immunoprecipitation followed by copy number array application (MeDIP-chip) for detecting methylation alterations. NspI restriction fragments were bound to anti-5-methylcytosine antibody, eluted, and hybridized to a Affymetrix Cytoscan HD probe. An unenriched total input fraction was processed for comparison. b Short hairpin mediated CTCF knockdown in two separate shRNA targeting CTCF verified by western blotting after 3 and 5 days of shRNA induction including shRNA non-silencing control (shNSC). Data shown are one representative of 3 independent experiments using immortalized HPECs. Percentage knockdown compared to shCTCF -Dox control, quantified by ImageJ. c Volcano plot of detected methylation changes in CTCF knockdown HPECE6/E7 after 5 days of dox exposure (cut-point, methylation Abs. Log2FC > 1.5, P

    Article Snippet: CTCF shRNA and controls were analyzed by western blotting using anti-CTCF rabbit monoclonal antibody (Cell Signaling #3418) to verify knockdown.

    Techniques: Methylation, Immunoprecipitation, Methylated DNA Immunoprecipitation, Chromatin Immunoprecipitation, shRNA, Western Blot

    Foxg1Cre -mediated deletion of Ctcf results in a massive increase in apoptosis. A , Table of genotypes obtained during the embryonic and postnatal periods. Ratios at each time point were analyzed using a χ 2 test. Het, Ctcf flox/WT ;Foxg1-cre +/− . B , Dark-field images of control and Ctcf Foxg1-cre embryos at E13.5. (Please note limbs were taken for genotyping). C , H E staining of E13.5 sagittal cryosections demonstrates complete loss of cortex (Ctx), hippocampal hem (H), basal ganglia (BG), lens (L), and anterior retina (AR), but not the posterior retina (PR), in Ctcf Foxg1-cre embryos. D , Dark-field images of control and Ctcf Foxg1-cre embryos at E11.5. The dashed circle outlines the telencephalon, which is visibly reduced in size in the Ctcf Foxg1-cre embryos compared to littermate controls. E , Immunodetection of CTCF (red) in E11.5 sagittal cryosections confirms specific loss of CTCF expression in the forebrain neuroepithelium of Ctcf Foxg1-cre embryos. F , Pregnant females were subjected to a 1 h BrdU pulse before being killed. Immunodetection of BrdU in E11.5 control and Ctcf Foxg1-cre cortical neuroepithelium is shown. G , BrdU + cells were counted and expressed as a percentage of the total number of DAPI + cells ( n = 3). H , Immunodetection of activated caspase-3 (red) in control and Ctcf Foxg1-cre cortical neuroepithelium at E11.5. I , AC3 + cells were counted and expressed as a percentage of the total number of DAPI + cells ( n = 3). J , TUNEL (green) detection in E11.5 control and Ctcf Foxg1-cre cortical neuroepithelium. Error bars represent the SEM. Original magnification: C , 25×; E , 50×; F , H , J , 200×. Scale bars: C , top, 1 mm; bottom, 400 μm; E , 200 μm; F , H , 50 μm; J , 100 μm.

    Journal: The Journal of Neuroscience

    Article Title: Dual Effect of CTCF Loss on Neuroprogenitor Differentiation and Survival

    doi: 10.1523/JNEUROSCI.3769-13.2014

    Figure Lengend Snippet: Foxg1Cre -mediated deletion of Ctcf results in a massive increase in apoptosis. A , Table of genotypes obtained during the embryonic and postnatal periods. Ratios at each time point were analyzed using a χ 2 test. Het, Ctcf flox/WT ;Foxg1-cre +/− . B , Dark-field images of control and Ctcf Foxg1-cre embryos at E13.5. (Please note limbs were taken for genotyping). C , H E staining of E13.5 sagittal cryosections demonstrates complete loss of cortex (Ctx), hippocampal hem (H), basal ganglia (BG), lens (L), and anterior retina (AR), but not the posterior retina (PR), in Ctcf Foxg1-cre embryos. D , Dark-field images of control and Ctcf Foxg1-cre embryos at E11.5. The dashed circle outlines the telencephalon, which is visibly reduced in size in the Ctcf Foxg1-cre embryos compared to littermate controls. E , Immunodetection of CTCF (red) in E11.5 sagittal cryosections confirms specific loss of CTCF expression in the forebrain neuroepithelium of Ctcf Foxg1-cre embryos. F , Pregnant females were subjected to a 1 h BrdU pulse before being killed. Immunodetection of BrdU in E11.5 control and Ctcf Foxg1-cre cortical neuroepithelium is shown. G , BrdU + cells were counted and expressed as a percentage of the total number of DAPI + cells ( n = 3). H , Immunodetection of activated caspase-3 (red) in control and Ctcf Foxg1-cre cortical neuroepithelium at E11.5. I , AC3 + cells were counted and expressed as a percentage of the total number of DAPI + cells ( n = 3). J , TUNEL (green) detection in E11.5 control and Ctcf Foxg1-cre cortical neuroepithelium. Error bars represent the SEM. Original magnification: C , 25×; E , 50×; F , H , J , 200×. Scale bars: C , top, 1 mm; bottom, 400 μm; E , 200 μm; F , H , 50 μm; J , 100 μm.

    Article Snippet: Primary antibodies used were as follows: rabbit anti-CTCF (1:400; Cell Signaling Technology), rabbit anti-cleaved caspase 3 (AC3; Asp175; 1:400; Cell Signaling Technology), mouse anti-BrdU (1:50; BD Biosciences), rabbit anti-PUMA (1:200; Cell Signaling Technology), rabbit anti-TBR2 (1:200; Abcam), goat anti-SOX2 (1:400; Santa Cruz Biotechnology), goat anti-PAX6 (1:200; Santa Cruz Biotechnology), rabbit anti-SOX2 (1:100; Millipore Bioscience Research Reagents), rabbit anti-Ki67 (1:200; Abcam), rabbit anti-TBR1 (1:200; Abcam), mouse anti-SATB2 (1:200; Abcam), and rabbit anti-CTIP2 (1:200; Abcam).

    Techniques: Staining, Immunodetection, Expressing, TUNEL Assay

    NestinCre -mediated deletion of CTCF results in activation of caspase-mediated apoptosis. A , Table of genotype ratios obtained during the embryonic period. Ratios at each time point were analyzed by a χ 2 test. B , Immunodetection of CTCF in E12.5 control and Ctcf Nes-cre coronal forebrain sections. C , Immunodetection of CTCF in E14 control and Ctcf Nes-cre coronal forebrain sections. D , Quantification of AC3 immunostaining in E12.5, E14, and E15.5 forebrain tissue ( n = 3). AC3 + cells were counted and expressed per unit area (square millimeter). E , Immunodetection of AC3 in E15.5 control and Ctcf Nes-cre basal ganglia. F , Immunodetection of AC3 in control and Ctcf Nes-cre at 4 DIV. Het, Ctcf flox/WT ;Nestin-cre ; Ctx, cortex; BG, basal ganglia; H, hippocampal hem. Error bars represent the SEM. Original magnification: B , C , 50×; D , 100×; E , 200×. Scale bars: B , 220 μm; C , 300 μm; E , 100 μm; F , 25 μm.

    Journal: The Journal of Neuroscience

    Article Title: Dual Effect of CTCF Loss on Neuroprogenitor Differentiation and Survival

    doi: 10.1523/JNEUROSCI.3769-13.2014

    Figure Lengend Snippet: NestinCre -mediated deletion of CTCF results in activation of caspase-mediated apoptosis. A , Table of genotype ratios obtained during the embryonic period. Ratios at each time point were analyzed by a χ 2 test. B , Immunodetection of CTCF in E12.5 control and Ctcf Nes-cre coronal forebrain sections. C , Immunodetection of CTCF in E14 control and Ctcf Nes-cre coronal forebrain sections. D , Quantification of AC3 immunostaining in E12.5, E14, and E15.5 forebrain tissue ( n = 3). AC3 + cells were counted and expressed per unit area (square millimeter). E , Immunodetection of AC3 in E15.5 control and Ctcf Nes-cre basal ganglia. F , Immunodetection of AC3 in control and Ctcf Nes-cre at 4 DIV. Het, Ctcf flox/WT ;Nestin-cre ; Ctx, cortex; BG, basal ganglia; H, hippocampal hem. Error bars represent the SEM. Original magnification: B , C , 50×; D , 100×; E , 200×. Scale bars: B , 220 μm; C , 300 μm; E , 100 μm; F , 25 μm.

    Article Snippet: Primary antibodies used were as follows: rabbit anti-CTCF (1:400; Cell Signaling Technology), rabbit anti-cleaved caspase 3 (AC3; Asp175; 1:400; Cell Signaling Technology), mouse anti-BrdU (1:50; BD Biosciences), rabbit anti-PUMA (1:200; Cell Signaling Technology), rabbit anti-TBR2 (1:200; Abcam), goat anti-SOX2 (1:400; Santa Cruz Biotechnology), goat anti-PAX6 (1:200; Santa Cruz Biotechnology), rabbit anti-SOX2 (1:100; Millipore Bioscience Research Reagents), rabbit anti-Ki67 (1:200; Abcam), rabbit anti-TBR1 (1:200; Abcam), mouse anti-SATB2 (1:200; Abcam), and rabbit anti-CTIP2 (1:200; Abcam).

    Techniques: Activation Assay, Immunodetection, Immunostaining

    Analysis of the rs4919742-associated chromatin loops. Guide RNAs targeting regions encompassing CTCF site 4 (the PCa risk-associated CTCF site), CTCF site 5, and/or CTCF site 6 (or the empty guide RNA vector as a control) were introduced into 22Rv1 prostate cancer cells, along with Cas9. Cell pools were harvested, and KRT78 expression was analyzed by RT-qPCR. Shown within the blue bars is the fold change in KRT78 expression in the pools that received guide RNAs vs the vector control. The yellow X indicates which CTCF site has been deleted; the size of each deletion can be found in Additional file 5 : Table S4

    Journal: Genome Biology

    Article Title: CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops

    doi: 10.1186/s13059-018-1531-0

    Figure Lengend Snippet: Analysis of the rs4919742-associated chromatin loops. Guide RNAs targeting regions encompassing CTCF site 4 (the PCa risk-associated CTCF site), CTCF site 5, and/or CTCF site 6 (or the empty guide RNA vector as a control) were introduced into 22Rv1 prostate cancer cells, along with Cas9. Cell pools were harvested, and KRT78 expression was analyzed by RT-qPCR. Shown within the blue bars is the fold change in KRT78 expression in the pools that received guide RNAs vs the vector control. The yellow X indicates which CTCF site has been deleted; the size of each deletion can be found in Additional file 5 : Table S4

    Article Snippet: Five micrograms of CTCF antibody (Active Motif #61311) was used to precipitate 20 μg chromatin for 22Rv1, PrEC, RWPE-2, VCaP (rep1) cells, and 10 ul CTCF antibody (Cell Signaling #3418S) were used to precipitate 20 μg chromatin for LNCaP, C4-2B, RWPE-1, VCaP (rep2) cells.

    Techniques: Plasmid Preparation, Expressing, Quantitative RT-PCR

    Experimental workflow for functional investigation of PCa risk-associated CTCF sites. Phase 1: Plasmids encoding guide RNAs that target sequences on each side of a PCa risk-associated CTCF site were introduced into the PCa cell line 22Rv1 along with a Cas9 expression vector (see the “ Methods ” section for details). The resultant cell pool was analyzed to determine deletion efficiency (red slashes represent alleles in each cell that harbor a CTCF site deletion). Single cells were then selected and expanded into clonal populations for RNA-seq analysis. Phase 2: After identifying the gene most responsive (within a ± 1-Mb window) to deletion of the region encompassing a risk-associated CTCF site, plasmids encoding guide RNAs that target the risk-associated CTCF anchor region and/or the regions encompassing the CTCF sites looped to the risk CTCF site and a Cas9 expression plasmid were introduced into 22Rv1 cells; cell pools were analyzed by PCR to check deletion frequency and by RT-qPCR to measure expression of the target gene

    Journal: Genome Biology

    Article Title: CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops

    doi: 10.1186/s13059-018-1531-0

    Figure Lengend Snippet: Experimental workflow for functional investigation of PCa risk-associated CTCF sites. Phase 1: Plasmids encoding guide RNAs that target sequences on each side of a PCa risk-associated CTCF site were introduced into the PCa cell line 22Rv1 along with a Cas9 expression vector (see the “ Methods ” section for details). The resultant cell pool was analyzed to determine deletion efficiency (red slashes represent alleles in each cell that harbor a CTCF site deletion). Single cells were then selected and expanded into clonal populations for RNA-seq analysis. Phase 2: After identifying the gene most responsive (within a ± 1-Mb window) to deletion of the region encompassing a risk-associated CTCF site, plasmids encoding guide RNAs that target the risk-associated CTCF anchor region and/or the regions encompassing the CTCF sites looped to the risk CTCF site and a Cas9 expression plasmid were introduced into 22Rv1 cells; cell pools were analyzed by PCR to check deletion frequency and by RT-qPCR to measure expression of the target gene

    Article Snippet: Five micrograms of CTCF antibody (Active Motif #61311) was used to precipitate 20 μg chromatin for 22Rv1, PrEC, RWPE-2, VCaP (rep1) cells, and 10 ul CTCF antibody (Cell Signaling #3418S) were used to precipitate 20 μg chromatin for LNCaP, C4-2B, RWPE-1, VCaP (rep2) cells.

    Techniques: Functional Assay, Expressing, Plasmid Preparation, RNA Sequencing Assay, Polymerase Chain Reaction, Quantitative RT-PCR

    Analysis of the rs12144978-associated chromatin loops. Guide RNAs targeting regions encompassing CTCF site 1 (the PCa risk-associated CTCF site), CTCF site 2, and/or CTCF site 3 (or the empty guide RNA vector as a control) were introduced into 22Rv1 prostate cancer cells, along with Cas9. Cell pools were harvested, and KCNN3 expression was analyzed by RT-qPCR. Shown within the blue bars is the fold change in KCNN3 expression in the pools that received guide RNAs vs. the vector control. The yellow X indicates which CTCF site has been deleted; the size of each deletion can be found in Additional file 5 : Table S4

    Journal: Genome Biology

    Article Title: CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops

    doi: 10.1186/s13059-018-1531-0

    Figure Lengend Snippet: Analysis of the rs12144978-associated chromatin loops. Guide RNAs targeting regions encompassing CTCF site 1 (the PCa risk-associated CTCF site), CTCF site 2, and/or CTCF site 3 (or the empty guide RNA vector as a control) were introduced into 22Rv1 prostate cancer cells, along with Cas9. Cell pools were harvested, and KCNN3 expression was analyzed by RT-qPCR. Shown within the blue bars is the fold change in KCNN3 expression in the pools that received guide RNAs vs. the vector control. The yellow X indicates which CTCF site has been deleted; the size of each deletion can be found in Additional file 5 : Table S4

    Article Snippet: Five micrograms of CTCF antibody (Active Motif #61311) was used to precipitate 20 μg chromatin for 22Rv1, PrEC, RWPE-2, VCaP (rep1) cells, and 10 ul CTCF antibody (Cell Signaling #3418S) were used to precipitate 20 μg chromatin for LNCaP, C4-2B, RWPE-1, VCaP (rep2) cells.

    Techniques: Plasmid Preparation, Expressing, Quantitative RT-PCR

    Experimental and analytical steps used to identify PCa risk-associated regulatory elements involved in chromatin loops. Step (1): The subset of 2,181 fine-mapped PCa-associated SNPs that overlap a DNase hypersensitive site was identified. Step (2): H3K27Ac and CTCF ChIP-seq was performed in duplicate in two normal (PrEC and RWPE-1) and five cancer (RWPE-2, 22Rv1, C4-2B, LNCaP, and VCaP) prostate cell lines; data was collected plus or minus DHT for 22Rv1 and LNCaP cells, for a total of 18 datasets for each mark (36 ChIP-seq samples). The SNPs in open chromatin sites (i.e., those that are contained within a DHS site) were then subdivided into those that overlap a H3K27Ac or a CTCF site in prostate cells; the number of PCa-associated SNPs associated with the H3K27Ac or CTCF sites is shown. Step (3): The PCa risk-associated H3K27Ac and CTCF sites were overlapped with Hi-C looping data, and the subset of each type of site involved in chromatin loops was identified; the number of PCa-associated SNPs associated with the H3K27Ac or CTCF sites involved in looping is shown

    Journal: Genome Biology

    Article Title: CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops

    doi: 10.1186/s13059-018-1531-0

    Figure Lengend Snippet: Experimental and analytical steps used to identify PCa risk-associated regulatory elements involved in chromatin loops. Step (1): The subset of 2,181 fine-mapped PCa-associated SNPs that overlap a DNase hypersensitive site was identified. Step (2): H3K27Ac and CTCF ChIP-seq was performed in duplicate in two normal (PrEC and RWPE-1) and five cancer (RWPE-2, 22Rv1, C4-2B, LNCaP, and VCaP) prostate cell lines; data was collected plus or minus DHT for 22Rv1 and LNCaP cells, for a total of 18 datasets for each mark (36 ChIP-seq samples). The SNPs in open chromatin sites (i.e., those that are contained within a DHS site) were then subdivided into those that overlap a H3K27Ac or a CTCF site in prostate cells; the number of PCa-associated SNPs associated with the H3K27Ac or CTCF sites is shown. Step (3): The PCa risk-associated H3K27Ac and CTCF sites were overlapped with Hi-C looping data, and the subset of each type of site involved in chromatin loops was identified; the number of PCa-associated SNPs associated with the H3K27Ac or CTCF sites involved in looping is shown

    Article Snippet: Five micrograms of CTCF antibody (Active Motif #61311) was used to precipitate 20 μg chromatin for 22Rv1, PrEC, RWPE-2, VCaP (rep1) cells, and 10 ul CTCF antibody (Cell Signaling #3418S) were used to precipitate 20 μg chromatin for LNCaP, C4-2B, RWPE-1, VCaP (rep2) cells.

    Techniques: Chromatin Immunoprecipitation, Hi-C

    Identification and classification of H3K27Ac ( a ) and CTCF ( b ) sites in prostate cells. H3K27Ac and CTCF ChIP-seq was performed in duplicate for each cell line; for 22Rv1 and LNCaP cells, ChIP-seq was performed in duplicate in the presence or absence of DHT. Peaks were called for individual datasets using MACS2 and the ENCODE3 pipeline, then peaks present in both replicates were identified (high confidence peaks) and used for further analysis (see Additional file 3 : Table S2). The location of the peaks was classified using the HOMER annotatePeaks.pl program and the Gencode V19 database. The fraction of high confidence peaks in each category is shown on the Y axis, with the number of peaks in each category for each individual cell line and/or treatment shown within each bar

    Journal: Genome Biology

    Article Title: CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops

    doi: 10.1186/s13059-018-1531-0

    Figure Lengend Snippet: Identification and classification of H3K27Ac ( a ) and CTCF ( b ) sites in prostate cells. H3K27Ac and CTCF ChIP-seq was performed in duplicate for each cell line; for 22Rv1 and LNCaP cells, ChIP-seq was performed in duplicate in the presence or absence of DHT. Peaks were called for individual datasets using MACS2 and the ENCODE3 pipeline, then peaks present in both replicates were identified (high confidence peaks) and used for further analysis (see Additional file 3 : Table S2). The location of the peaks was classified using the HOMER annotatePeaks.pl program and the Gencode V19 database. The fraction of high confidence peaks in each category is shown on the Y axis, with the number of peaks in each category for each individual cell line and/or treatment shown within each bar

    Article Snippet: Five micrograms of CTCF antibody (Active Motif #61311) was used to precipitate 20 μg chromatin for 22Rv1, PrEC, RWPE-2, VCaP (rep1) cells, and 10 ul CTCF antibody (Cell Signaling #3418S) were used to precipitate 20 μg chromatin for LNCaP, C4-2B, RWPE-1, VCaP (rep2) cells.

    Techniques: Chromatin Immunoprecipitation

    PCa risk SNPs associated with CTCF sites and chromatin loops. Each row represents one of the 93 SNPs that are associated with both a DHS site and a CTCF peak in normal or tumor prostate cells (Additional file 4 : Table S3). The location of each SNP was classified using the Gencode V19 database. “Others” represents mostly intergenic regions. To identify the subset of CTCF-associated risk SNPs located in an anchor point of a loop, chromatin loops were identified using Hi-C data from normal RWPE-1 prostate cells [ 26 ] or 22Rv1 and C4-2B prostate tumor cells (Rhie et al., in preparation); Hi-C [ 25 ] and cohesin HiChIP data [ 27 ] from GM12878 was also used

    Journal: Genome Biology

    Article Title: CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops

    doi: 10.1186/s13059-018-1531-0

    Figure Lengend Snippet: PCa risk SNPs associated with CTCF sites and chromatin loops. Each row represents one of the 93 SNPs that are associated with both a DHS site and a CTCF peak in normal or tumor prostate cells (Additional file 4 : Table S3). The location of each SNP was classified using the Gencode V19 database. “Others” represents mostly intergenic regions. To identify the subset of CTCF-associated risk SNPs located in an anchor point of a loop, chromatin loops were identified using Hi-C data from normal RWPE-1 prostate cells [ 26 ] or 22Rv1 and C4-2B prostate tumor cells (Rhie et al., in preparation); Hi-C [ 25 ] and cohesin HiChIP data [ 27 ] from GM12878 was also used

    Article Snippet: Five micrograms of CTCF antibody (Active Motif #61311) was used to precipitate 20 μg chromatin for 22Rv1, PrEC, RWPE-2, VCaP (rep1) cells, and 10 ul CTCF antibody (Cell Signaling #3418S) were used to precipitate 20 μg chromatin for LNCaP, C4-2B, RWPE-1, VCaP (rep2) cells.

    Techniques: Hi-C, HiChIP

    Topology of the EGFR locus in unamplified and amplified glioblastoma. ( a ) (Top) HiC contact map of unamplified glioblastoma model cell line G567. (Middle) CTCF ChlP-seq track of unamplified glioblastoma model cell line GSC23. The red and green arrows denote the orientation of the CTCF motif. (Bottom) Schematic of the boundaries of the loop domain. ( b ) 4C-seq profiles anchored from near the EGFR transcription start site (dotted line) showing interactions to the EGFR enhancers (boxed) for GSC23 (unamplified). Red arcs denote regions with strong distal interactions with EGFR at a 12 kb window thresholded at a 0.12 interaction frequency. H3K27ac ChIP-seq tracks accompany the 4C-seq data. The 4C-seq tracks consist of a main trend of the chromatin interactions based on a 5 kb window and domainogram colored by interaction frequency. Right side depicts a model for the toplogy of the locus. ( c ) Same as (b) for GBM3565 ( EGFR .

    Journal: Cell

    Article Title: Functional enhancers shape extrachromosomal oncogene amplifications

    doi: 10.1016/j.cell.2019.10.039

    Figure Lengend Snippet: Topology of the EGFR locus in unamplified and amplified glioblastoma. ( a ) (Top) HiC contact map of unamplified glioblastoma model cell line G567. (Middle) CTCF ChlP-seq track of unamplified glioblastoma model cell line GSC23. The red and green arrows denote the orientation of the CTCF motif. (Bottom) Schematic of the boundaries of the loop domain. ( b ) 4C-seq profiles anchored from near the EGFR transcription start site (dotted line) showing interactions to the EGFR enhancers (boxed) for GSC23 (unamplified). Red arcs denote regions with strong distal interactions with EGFR at a 12 kb window thresholded at a 0.12 interaction frequency. H3K27ac ChIP-seq tracks accompany the 4C-seq data. The 4C-seq tracks consist of a main trend of the chromatin interactions based on a 5 kb window and domainogram colored by interaction frequency. Right side depicts a model for the toplogy of the locus. ( c ) Same as (b) for GBM3565 ( EGFR .

    Article Snippet: Samples were sheared in 1 ml milliTUBEs on the Covaris model S2 AFA focused ultrasonicator, duty factor 2%, intensity 4, 200 cycles/burst, for 6 min. Our ChlP-seq method using 8 μg rabbit anti-H3K27ac (Abeam #4729) or 10 μL of anti-CTCF (Cell Signaling #3418) was that of Schmidt et al. ( ) but using PCRCIean Dx paramagnetic beads (ALINE #C-1003) instead of column purifications and using adapters that we made following Illumina bar codes.

    Techniques: Amplification, Chromatin Immunoprecipitation