Identification of transcription factor binding sites from ChIP-seq data at high-resolution
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ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE40664 | GEO | 2014/02/06
SECONDARY ACCESSION(S): PRJNA174520
REPOSITORIES: GEO
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Project description:Genome-wide mapping of proteinM-bM-^@M-^SDNA interactions is essential for a full understanding of transcriptional regulation. A precise map of binding sites for transcription factors, core transcriptional machinery is vital for deciphering the gene regulatory networks that underlie various biological processes. Chromatin immunoprecipitation followed by sequencing (ChIPM-bM-^@M-^Sseq) is a technique for genome-wide profiling of DNA-binding proteins. However, our conventional ChIPM-bM-^@M-^Sseq occasionally gives wider peaks which might be due to overlapping binding sites of two or more transcription factors. Therefore, to improve the resolution of our conventional ChIPM-bM-^@M-^Sseq which have DNA-protein footprint of ~100 bp, we decreased the size of DNA-protein footprint to ~ 50 bp by DNaseI digestion of whole cell extract (WCE). ChIP-seq for Twist transcription factor in Drosophila embryos
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Project description:This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Richard Sandstrom mailto:sull@u.washington.edu). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu). This track, produced as part of the mouse ENCODE Project, contains deep sequencing DNase data that will be used to identify sites where regulatory factors bind to the genome (footprints). Footprinting is a technique used to define the DNA sequences that interact with and bind DNA-binding proteins, such as transcription factors, zinc-finger proteins, hormone-receptor complexes, and other chromatin-modulating factors like CTCF. The technique depends upon the strength and tight nature of protein-DNA interactions. In their native chromatin state, DNA sequences that interact directly with DNA-binding proteins are relatively protected from DNA degrading endonucleases, while the exposed/unbound portions are readily degraded by such endonucleases. A massively parallel next-generation sequencing technique to define the DNase hypersensitive sites in the genome was adopted. The DNase samples were sequenced using next-generation sequencing machines to significantly higher depths of 300-fold or greater. This produces a base-pair level resolution of the DNase susceptibility maps of the native chromatin state. These base-pair resolution maps represent and are dependent upon the nature and the specificity of interaction of the DNA with the regulatory/modulatory proteins binding at specific loci in the genome; thus they represent the native chromatin state of the genome under investigation. The deep sequencing approach has been used to define the footprint landscape of the genome by identifying DNA motifs that interact with known or novel DNA binding proteins. Cells were grown according to the approved ENCODE cell culture protocols (http://hgwdev.cse.ucsc.edu/ENCODE/protocols/cell/mouse). Digital DNaseI was performed by DNaseI digestion of intact nuclei, followed by isolating DNaseI "double-hit" fragments (Sabo et al., 2006), and direct sequencing of fragment ends (which correspond to in vivo DNaseI cleavage sites) using the Solexa platform (27 bp reads). High-quality reads were mapped to the NCBI37/mm9 mouse genome using Bowtie 0.12.5; only unique mappings were kept. DNaseI sensitivity is directly reflected in raw tag density (Raw Signal), which is shown in the track as density of tags mapping within a 150 bp sliding window (at a 20 bp step across the genome). DNaseI hypersensitive zones (HotSpots) were identified using the HotSpot algorithm (Sabo et al., 2004). False discovery rate thresholds of 1.0% (FDR 0.01) were computed for each cell type by applying the HotSpot algorithm to an equivalent number of random uniquely mapping 36-mers. DNaseI hypersensitive sites (DHSs or Peaks) were identified as signal peaks within 1.0% (FDR 0.01) hypersensitive zones using a peak-finding algorithm. Only DNase Solexa libraries from unique cell types producing the highest quality data, as defined by Percent Tags in Hotspots (PTIH ~40%), were designated for deep sequencing to a depth of over 200 million tags. Results were validated by conventional DNaseI hypersensitivity assays using end-labeling/Southern blotting methods.
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