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Genome-wide binding profile of PcG proteins Pho and Ph, H3K27me3 and Engrailed in Drosophila third instar larval brains and disks, in duplicate, using Illumina HiSeq 2500. The sequence reads that passed quality filters were analyzed. Drosophila third instar larval brains and disks were dissected and fixed with 2% HCHO before sonication. After sonication, chromatin immunoprecipitation using anti-Pho, -Ph, -H3K27me3, and -Engrailed antibodies was carried out.
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Genome-wide binding profile of PcG proteins- Ph, H3K27me3 and Cg in Drosophila third instar larval brains and disks, in duplicate, using Illumina HiSeq 2500. The sequence reads that passed quality filters were analyzed. Drosophila third instar larval brains and discs were dissected and fixed with 2% HCHO before sonication. After sonication, chromatin immunoprecipitation was carried out.
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modENCODE_submission_4192 This submission comes from a modENCODE project of David MacAlpine. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We will precisely identify sequence elements that direct DNA replication by using chromatin immunoprecipitation of known replication initiation complexes. These experiments will be conducted in multiple cell types and developmental tissues. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Strain: Oregon-R(official name : Oregon-R-modENCODE genotype : wild type ); Developmental Stage: Embryo 4-7h; Genotype: wild type; EXPERIMENTAL FACTORS: Strain Oregon-R(official name : Oregon-R-modENCODE genotype : wild type ); read length (read_length) ; Antibody dORC2 (target is Drosophila ORC2p); Developmental Stage Embryo 4-7h
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Sequence-specific transcription factors (TFs) are critical for specifying patterns and levels of gene expression, but the DNA elements to which they can bind are not always sufficient to specify their binding in vivo. In eukaryotes, the binding of a TF is in competition with a constellation of other proteins, including histones which package DNA into nucleosomes. Here, we examine using the ChIP-seq assay, the genome-wide distribution of Drosophila Heat Shock Factor (HSF), a TF whose binding activity is mediated by heat shock-induced trimerization. We detect HSF binding to 464 sites, the vast majority of which contain HSF Sequence-binding Elements (HSEs) in Drosophila S2 cells, but these HSF-bound sites represent only a small fraction of HSEs present in the genome. We find a strong correlation of bound HSEs to active chromatin marks present prior to HSF binding, indicating an HSE’s residence in open chromatin is a primary determinant of whether HSF can bind following heat shock. A single mock immunoprecipitation (IP) using the pre-innoculated animal serum was used as a background dataset for this study. For each condition (NHS and 20minute HS), we performed two independent HSF-ChIP-seq experiments. In addition, we performed two independent HSF-ChIP-seq experiments for each condition (NHS and 20minute HS) in cells that were highly depleted of HSF by RNAi.
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Chromatin immunoprecipitation using antibodies against pMad on 2 to 2.5 hr and 3 to 3.5 hr Drosophila embryos, detected by SOLiD sequencing
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We report here the genome-wide localization of the histone acetyltransferase MYST5 (cg1984) in Drosophila. ChIP-seq analysis was performed with two anti-MYST5 antibodies in S2 cells. We found MYST5 to bind to the promoters of actively transcribed genes. MYST5 furthermore showed extensive colocalization with boundary/ insulator factors, including Chriz/ Chromator, CP190, dCTCF and BEAF-32, which mediate the organization of the genome into functionally distinct topological domains. Altogether, our data suggest a broad role for MYST5 both in gene-specific transcriptional regulation and in the organization of the genome into chromatin domains. Examination of genome-wide MYST5 localization in S2 cells
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Here we examine changes in the distribution of Drosophila insulator proteins during the ecdysone response. We performed ChIP-seq analysis in Kc cells at 0, 3, and 48 hours of ecdysone treatment with antibodies against CP190, Su(Hw), dCTCF, and BEAF-32B. Examination of 4 different insulator proteins at 3 time points of ecdysone treatment.
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We analyzed ORC2 ChIP-Seq from hand dissected salivary glands of wandering third instar larvae from OrR or SuUR Drosophila. Goals were to ascertain the difference in binding profile between salivary glands expressing and not expressing the Supressor of UnderReplication protein. One replicate is included for each of OrR (WT) or SuUR salivary glands.
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Chromatin immunoprecipitation using antibodies against Brk on 2 to 2.5 hr and 3 to 3.5 hr Drosophila embryos, detected by SOLiD sequencing
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DNA sequence and local chromatin landscape act jointly to determine transcription factor (TF) binding intensity profiles. To disentangle these influences, we developed an experimental approach, called protein/DNA binding and high-throughput sequencing (PB-seq), that allows the binding energy landscape to be characterized genome-wide in the absence of chromatin. We applied our methods to the Drosophila Heat Shock Factor (HSF), which inducibly binds a target DNA sequence element (HSE) following heat shock stress. PB-seq involves incubating sheared naked genomic DNA with recombinant HSF, partitioning the HSF-bound and HSF-free DNA, and then detecting HSF-bound DNA by high throughput sequencing. We compared PB-seq binding profiles with ones observed in vivo by ChIP-seq, and developed statistical models to predict the observed departures from idealized binding patterns based on covariates describing the local chromatin environment. We found that DNase I hypersensitivity and tetra-acetylation of H4 were the most influential covariates in predicting changes in HSF binding affinity. We also investigated the extent to which DNA accessibility, as measured by digital DNase I footprinting data, could be predicted from MNase-seq data and the ChIP-chip profiles for many histone modifications and TFs, and found GAGA element associated factor (GAF), tetra-acetylation of H4, and H4K16 acetylation to be the most predictive covariates. Lastly, we generated an unbiased model of HSF binding sequences, which revealed distinct biophysical properties of the HSF/HSE interaction and a previously unrecognized substructure within the HSE. These findings provide new insights into the interplay between the genomic sequence and the chromatin landscape in determining transcription factor binding intensity. We performed an in vitro binding experiment with purified HSF and naked, sheared genomic Drosophila S2 DNA (PB-seq), to derive an accurate set of potential HSF binding sites in the Drosophila genome. HSF-bound DNA was specifically eluted and detected by high throughput sequencing. Drosophila HSF was N-terminally tagged with glutathione s-transferase and a tobacco etch virus (TEV) protease cleavage site. The C-terminus of the recombinant HSF was fused to the 3xFLAG epitope. Recombinant HSF was purified from E. coli with glutathione resin as previously described (PMID: 20078429) , with the following modifications: HSF-3xFLAG elution was achieved by addition of 6xHistidine tagged TEV protease and TEV protease was cleared from the HSF preparation using a Nickel-NTA column. We incubated 600pM HSF and 2500ng genomic DNA (sonicated to 100-600bp fragment size as previously described in PMID: 20844575) in 1500μl final volume of 1xHSF binding buffer and let it come to equilibrium for an hour at room temperature. We added 20μl ANTI-FLAG M2 affinity gel for 10 minutes, washed 8 times with 1xHSF binding buffer to remove unbound DNA; 3xFLAG peptide was added to a final concentration of 200ng/μl to specifically elute HSF and HSF-bound DNA. The mock IP was done in the absence of recombinant HSF.
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