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H3-ChIP-seq was performed in order to analyze changes in nucleosomal occupancy after depletion of CTCF/P190 and ISWI from Drosophila S2 cells Histone H3 ChIP-seq from Drosophila S2 cells after CTCF/CP190 or ISWI-specific RNAi treatment
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Accession Number: GSE63323 Platform: GPL13304: Illumina HiSeq 2000 (Drosophila melanogaster) Organism: Drosophila melanogaster Published on 2014-11-21 Summary: Alternative polyadenylation (APA) has been implicated in a variety of developmental and disease processes, such as stem cell differentiation and cancer. A particularly dramatic form of APA has been documented in the developing nervous system of flies and mammals, whereby a variety of neurogenic genes undergo coordinate extension of their 3’ UTRs. In Drosophila, the RNA-binding protein ELAV inhibits RNA processing at proximal polyadenylation (poly(A)) sites, thereby fostering the formation of 3’ extensions that can reach 12 kb in length. Here, we present evidence that paused Pol II plays an important role in the selective recruitment of ELAV to elongated genes. Replacing native promoters of elongated genes with heterologous promoters blocks normal 3’ extension in the nervous system, while native promoters can induce 3’ extension in ectopic tissues expressing ELAV. Computational analyses suggest that the promoter regions of elongated genes tend to contain paused Pol II and associated cis-regulatory elements such as GAGA. ELAV ChIP-Seq assays indicate pervasive binding to the promoter regions of extended genes. Our study provides the first evidence for a regulatory link between promoter-proximal pausing and APA. Overall Design: ELAV ChIP-Seq assays were conducted with nuclei obtained from 6-8 hr and 10-12 hr embryos Contact: Name: Wei Zhang Organization: UC Berkeley Address: University of California at Berkeley Berkeley 94720 USA Email: wzhang1984@gmail.com Organization: GEO Address: USA
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Accession Number: GSE74413 Platform: GPL17275: Illumina HiSeq 2500 (Drosophila melanogaster) Organism: Drosophila melanogaster Published on 2016-08-02 Summary: The goal of this experiment was to identify genomic dCAP-D3 binding sites in Drosophila S2R+ cells Overall Design: dCAP-D3 was immunoprecipitated in two separate experiments with antibody YZ834 which was developed in the Longworth lab. IgG immunoprecipiation was performed alongside the dCAP-D3 immunoprecipitation to serve as controls for each experiment. Input chromatin was also harvested from each of the two experiments. Contact: Name: Michelle Suzanne Longworth Organization: Cleveland Clinic Foundation Laboratory: Longworth Deparment: Molecular Genetics Address: 9500 Euclid Ave NE20 Cleveland OH 44195 USA Email: longwom@ccf.org Phone: 216-618-6114 Organization: GEO Address: USA
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ChIP against Opbp followed by next generation sequencing of Drosophila melanogaster embryos. Samples were sequenced using Illumina HiSeq and include four biological replicates, with both mock and input controls.
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Alternative polyadenylation (APA) has been implicated in a variety of developmental and disease processes, such as stem cell differentiation and cancer. A particularly dramatic form of APA has been documented in the developing nervous system of flies and mammals, whereby a variety of neurogenic genes undergo coordinate extension of their 3’ UTRs. In Drosophila, the RNA-binding protein ELAV inhibits RNA processing at proximal polyadenylation (poly(A)) sites, thereby fostering the formation of 3’ extensions that can reach 12 kb in length. Here, we present evidence that paused Pol II plays an important role in the selective recruitment of ELAV to elongated genes. Replacing native promoters of elongated genes with heterologous promoters blocks normal 3’ extension in the nervous system, while native promoters can induce 3’ extension in ectopic tissues expressing ELAV. Computational analyses suggest that the promoter regions of elongated genes tend to contain paused Pol II and associated cis-regulatory elements such as GAGA. ELAV ChIP-Seq assays indicate pervasive binding to the promoter regions of extended genes. Our study provides the first evidence for a regulatory link between promoter-proximal pausing and APA. ELAV ChIP-Seq assays were conducted with nuclei obtained from 6-8 hr and 10-12 hr embryos
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Here we use ChIP-seq in Drosophila embryos to determine the genome-wide binding pattern of TBP and Trf2 using two different antibodies for each factor. ChIP-seq using anti-Trf2 and anti-TBP antibodies in Drosophila embryos
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The goal of this experiment was to identify genomic dCAP-D3 binding sites in Drosophila S2R+ cells dCAP-D3 was immunoprecipitated in two separate experiments with antibody YZ834 which was developed in the Longworth lab. IgG immunoprecipiation was performed alongside the dCAP-D3 immunoprecipitation to serve as controls for each experiment. Input chromatin was also harvested from each of the two experiments.
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ChIP-chip and ChIP-seq data analysis workflow. ChIP-chip data (fold enrichment of immunoprecipitated material over genomic DNA) and/or ChIP-seq data are mapped to a reference genome. Control bound and unbound regions are visually inspected and validated by comparison to standard ChIP and qPCR. Genomic regions where signal is significantly greater than expected by chance (user-defined threshold) are identified as ‘bound’. Bound regions are then compared to a database of genomic elements of interest (e.g. promoters) to identify bound elements. Note that absence of detected binding from a genomic region may result from absence of complementary probes upon the array (ChIP-chip), masking of repetitive regions (ChIP-chip and ChIP-seq), or unmappable regions (ChIP-seq). ... Pol II distribution detected by ChIP, ChIP-chip, and ChIP-seq. Drosophila S2 cells were crosslinked, sonicated, and total Pol II (Rpb3) was immunoprecipitated. Pol II ChIP signal at the Tl promoter region was quantified with qPCR using primer pairs spaced on average every 100bp (blue line), ChIP-chip using Agilent Drosophila Whole Genome 2-ChIP sets with average probe spacing of 250bp (red line), or ChIP-seq reads sequenced with the Illumina Genome Analyzer (green line), binned in 25 nucleotide windows. Genomic positions are reported as bp×10−2, and represent the center point of primer pairs used, probe sequence, or window. ... ChIP-seq... Comparison of Pol II distribution as determined by ChIP-chip and ChIP-seq. The distribution of Pol II (Rpb3)-binding at the: (A) lace; (B) kay; (C) smi35A and; (D) CG6860/CLIP-190 genes was determined by ChIP-chip (NimbleGen HD2 Drosophila whole genome arrays) or ChIP-seq (Illumina Genome Analyzer reads binned in 25 nucleotide windows). ... Workflow for ChIP-chip and ChIP-seq experiments. Following experimental manipulation (yellow boxes), cells are crosslinked with formaldehyde, sonicated to fragment chromatin, and protein–DNA complexes immunoprecipitated with antibodies targeting the protein or modification of interest (here, Pol II). Following quality control qPCR to confirm expected ChIP signal at control regions, immunoprecipitated DNA is processed specifically for either ChIP-chip or ChIP-seq. ChIP-chip can provide information about all immunoprecipitated DNA sequences complementary to tiling array probes in a strand-insensitive manner. ChIP-seq provides information about all mappable sequences located at the 5′-ends of immunoprecipitated DNA (red and blue boxes). ... Pol II binding detected with differing ChIP-chip methods and platforms. Pol II (Rpb3) ChIP was performed with material generated from Drosophila S2 cells and binding was detected with NimbleGen HD2 Drosophila whole genome arrays or Agilent Drosophila Whole Genome 2-ChIP sets. Pol II-bound genomic regions were determined for the NimbleGen array with NimbleScan software (FDRDrosophila Release 5 Genomic sequence. Pol II-bound genomic regions were determined for the Agilent arrays with the Drosophila Release 3 Genomic sequence as previously described [4,5,48].
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We analyzed gamaH2Av ChIP-seq from hand dissected stage 10B and 13 follicle cell nuclei. Egg chambers were dissected from wild-type (OrR) or H2Av[ΔCT] ovaries to assess binding at the Drosophila Follicle Cell Amplicons and across the genome. ChIP-seq of gammaH2Av bound to follicle cell DNA from stage 10B and 13 egg chambers, collected from wild-type (OrR) and H2Av[ΔCT] Drosophila ovaries. Sequences analyzed by Illumina sequencing. Two replicates are included for each ChIP reaction.
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Accession Number: GSE66689 Platform: GPL13304: Illumina HiSeq 2000 (Drosophila melanogaster) Organism: Drosophila melanogaster Published on 2015-06-18 Summary: We analyzed gamaH2Av ChIP-seq from hand dissected stage 10B and 13 follicle cell nuclei. Egg chambers were dissected from wild-type (OrR) or H2Av[ΔCT] ovaries to assess binding at the Drosophila Follicle Cell Amplicons and across the genome. Overall Design: ChIP-seq of gammaH2Av bound to follicle cell DNA from stage 10B and 13 egg chambers, collected from wild-type (OrR) and H2Av[ΔCT] Drosophila ovaries. Sequences analyzed by Illumina sequencing. Two replicates are included for each ChIP reaction. Contact: Name: Terry L. Orr-Weaver Organization: Whitehead Institute for Biomedical Research Laboratory: Orr-Weaver Address: 9 Cambridge Center Cambridge MA 02142 USA Email: weaver@wi.mit.edu Phone: 617-258-5251 Organization: GEO Address: USA
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