Genome binding/occupancy profiling by high throughput sequencing
Summary
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 was produced as part of the ENCODE Project. This track displays genome-wide maps of histone modifications in different cell lines (http://hgwdev.cse.ucsc.edu/cgi-bin/hgEncodeVocab?type=cellType) using ChIP-seq high-throughput sequencing.
Cells were grown according to the approved ENCODE cell culture protocols (http://hgwdev.cse.ucsc.edu/ENCODE/protocols/cell). Cells were cross-linked with 1% formaldehyde, and the reaction was quenched by the addition of glycine. Fixed cells were rinsed with PBS, lysed in nuclei lysis buffer, and the chromatin was sheared to 200-500 bp fragments using a Fisher Dismembrator (model 500). Sheared chromatin fragments were immunoprecipitated with specific polyclonal antibodies at 4 °C with gentle rotation. Antibody-chromatin complexes were washed and eluted. The cross-linking in the immunoprecipitated DNA was reversed and treated with RNase-A. Following proteinase K treatment, the DNA fragments were purified by phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation. A quantity of 20-50 ng of ChIP DNA was end-repaired, followed by the addition of adenine, ligation to Illumina adapters, and creation of a Solexa library for sequencing. ChIP-seq affinity was directly measured through the 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). ChIP-seq affinity zones (HotSpots) were identified using the HotSpot algorithm described in Sabo et al. (2004). One percent false discovery rate thresholds (FDR 1.0%) were computed for each cell type by applying the HotSpot algorithm to an equivalent number of random uniquely mapping 36-mers. ChIP-Seq affinities (Peaks) were identified as signal peaks within FDR 1.0% hypersensitive zones using a peak-finding algorithm. All tracks have a False Discovery Rate of 1% (FDR 1.0%). Data were verified by sequencing biological replicates displaying a correlation coefficient > 0.9.
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