Unknown,Transcriptomics,Genomics,Proteomics

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Transcription Factor Binding Sites by Epitope-Tag ChIP-seq from ENCODE/University of Chicago

ABSTRACT: This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Kevin White mailto:kpwhite@uchicago.edu (Principal Investigator), Subhradip Karmakar mailto:subhradip@uchicago.edu (Project Lead), Nick Bild mailto:nbild@bsd.uchicago.edu (Data Analyst), Alina Choudhury mailto:achoudhury@uchicago.edu (Laboratory Technician), Marc Domanus mailto:mdomanus@anl.gov (Sequencing Technician at Argonne National Lab)). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu). This ENCODE track maps human transcription factor binding sites, genome-wide using second generation massively parallel sequencing. This mapping uses expressed transcription factors as GFP tagged fusion proteins after BAC (Bacterial artificial chromosomes) recombineering. The U. of Chicago and Max Planck Institute (Dresden) pipeline generates recombineered (recombination-mediated genetic engineering) BACs for the production of cell lines or animals that express fusion proteins from epitope tagged transgenes. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Cells were grown according to the approved ENCODE cell culture protocols. (http://hgwdev/ENCODE/protocols/cell) Recombineering strategy: To facilitate high-throughput production of the transgenic constructs, the program BACFinder (Crowe, Rana et al. 2002) automatically selects the most suitable BAC clone for any given human gene and generates the sets of PCR primers required for tagging and verification (Poser, Sarov et al. 2008). Recombineering is used for tagging cassettes at either the N or C terminus of the protein. The N-terminal cassette has a dual eukaryotic-prokaryotic promoter (PGK-gb2) driving a neomycin-kanamycin resistance gene within an artificial intron inside the tag coding sequence. The selection cassette is flanked by two loxP sites and can be permanently removed by Cre recombinase-mediated excision. The C-terminal cassette contains the sequence encoding the tag followed by an internal ribosome entry site (IRES) in front of the neomycin resistance gene. In addition, a short bacterial promoter (Gb3) drives the expression of the neomycin-kanamycin resistance gene in E. coli. The tagging cassettes, containing 50 nucleotides of PCR-introduced homology arms are inserted into the BAC by recombineering, either behind the start codon (for the N-terminal tag) or in front of the stop codon (for the C-terminal tag) of the gene. E. coli cells that have successfully recombined the cassette are selected for kanamycin resistance in liquid culture. Each saturated culture from a specific recombineering reaction derived 10-200 independent recombination events. Checking two independent clones for each PCR through the tag insertion point, 97% (85/88) yielded a PCR product of the expected size. Most of the clones that failed to grow were missing the targeted genomic region. An estimated 10% of the BACs used are chimeric, rearranged or wrongly mapped. Thus, initial results indicate that the necessary recombineering steps can be carried out with high fidelity. The White lab produced all epitope tagged transcription and chromatin factor BACs, as well as the genome wide ChIP data and analysis. An application of this approach to the analysis of closely related paralogs (RARa and RARg) yielded transcription factors, chromatin factors, cell lines, ChIP chip data and ChIP-seq data (Hua, Kittler et al. Cell 2009). Such paralogous transcription factors often can not otherwise be distinguished by antibodies. Sample Preparation: ChIP DNA from samples are sheared to ~800bp using a nebulizer. The ends of the DNA are polished, and two unique adapters are ligated to the fragments. Ligated fragments of 150-200bp are isolated by gel extraction and amplified using limited cycles of PCR. Sequencing System: Illumina GAIIx and HySeq next-generation sequencing produced all ChIP-seq data. Processing and Analysis Software: Raw sequencing reads are aligned using Bowtie version 0.12.5 (Langmead et al. 2009). The "-m 1" parameter is applied to suppress alignments mapping more than once in the genome. Reads are aligned to the UCSC hg19 assembly. Wiggle format signal files are generated with SPP 2.7.1 for R 2.7.1. Macs 1.3.7 is used to call peaks. The Macs parameters used vary by experiment. The White lab used goat anti-GFP antibody to perform ChIP in untagged K562 cells as a background control. The test IP was performed in the same way as the background control. Results are expressed as values of the test normalized to the background

ORGANISM(S): Homo sapiens

SUBMITTER: UCSC ENCODE DCC

PROVIDER: E-GEOD-31363 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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 is produced as part of the ENCODE Project. This track displays maps of genome-wide binding of the CTCF transcription factor in different cell lines using ChIP-seq high-throughput sequencing For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Cells were grown according to the approved ENCODE cell culture protocols. Cells were crosslinked 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 Fisher Dismembrator (model 500). Sheared chromatin fragments were immunoprecipitated with specific polyclonal antibodies at 4 degrees C with gentle rotation. Antibody-chromatin complexes were washed and eluted. The cross linking in 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. 20-50 ng of ChIP DNA was end-repaired, adenine ligated to Illumina adapters was added, and then a Solexa library was made for sequencing. ChIP-seq affinity 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). ChIP-seq affinity zones (HotSpots) were identified using the HotSpot algorithm described in Sabo et al. (2004). 1.0% false discovery rate thresholds (FDR 0.01) were computed for each cell type by applying the HotSpot algorithm to an equivalent number of random uniquely mapping 36mers. ChIP-seq affinity (Peaks) were identified as signal peaks within FDR 1.0% hypersensitive zones using a peak-finding algorithm.

Project description:This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Yijun Ruan mailto:ruanyj@gis.a-star.edu.sg). 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. It shows the locations of protein factor mediated chromatin interactions determined by Chromatin Interaction Analysis with Paired-End Tag (ChIA-PET) data (Fullwood et al., 2010) extracted from five different human cancer cell lines (K562 (chronic myeloid leukemia), HCT116 (colorectal cancer), HeLa-S3 (cervical cancer), MCF-7 (breast cancer), and NB4 (promyelocytic)). A chromatin interaction is defined as the association of two regions of the genome that are far apart in terms of genomic distance, but are spatially proximate to each other in the 3-dimensional cellular nucleus. Additionally, ChIA-PET experiments generate transcription factor binding sites. A binding site is defined as a region of the genome that is highly enriched by specific Chromatin ImmunoPrecipitation (ChIP) against a transcription factor, which indicates that the transcription factor binds specifically to this region. The protein factors displayed in the track include estrogen receptor alpha (ERa), RNA polymerase II (RNAPII), and CCCTC binding factor (CTCF). For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Chromatin interaction analysis with paired-end tag sequencing (ChIA-PET) is a global de novo high-throughput method for characterizing the 3-dimensional structure of chromatin in the nucleus. In the ChIA-PET protocol, samples were cross-linked and fragmented, then subjected to chromatin immunoprecipitation. The DNA fragments that were brought together by the chromatin interactions were then proximity-ligated. During this proximity-ligation step, the half-linkers (created by the fragmentation) containing flanking MmeI sites (type IIS restriction enzymes) were first ligated to the DNA fragments and then ligated to each other to form full linkers. Full linkers bridge either two ends of a self-circularized fragment, or two ends of two different chromatin fragments. The material was then reverse cross-linked, purified and digested with MmeI. MmeI cuts 20 base pairs away from its recognition site. Tag-linker-tag (paired-end tag, PET) constructs were sequenced by ultra-high-throughput methods (Illumina or SOLiD paired-end sequencing). ChIA-PET reads were processed with the ChIA-PET Tool (Li et al., 2010) by the following steps: linker filtering, short reads mapping, PET classification, binding site identification, and interaction cluster identification. The high-confidence binding sites and chromatin interaction clusters were reported. Chromatin interactions identified by ChIA-PET have been validated by 3C, ChIP-3C, 4C and DNA-FISH (Fullwood et al., 2009).

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 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. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf 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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Transcription Factor Binding Sites by Epitope-Tag ChIP-seq from ENCODE/University of Chicago

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