Unknown,Transcriptomics,Genomics,Proteomics

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Characterization of technical variation in ChIP-on-chip studies

ABSTRACT: Analysis of technical variance of ChIP-on-Chip studies by characterization of Myc-binding in HL60 cells. Keywords: Chip-on-chip Fully-blocked study of technical variance in Myc-binding in HL60 cells. Two different antibodies were used to generate 6 biologically independent replicates each. Each replicate was hybridized to arrays from two different batches in both dye-swap orientations, leading to 48 total arrays.

ORGANISM(S): Homo sapiens

SUBMITTER: Paul Boutros

PROVIDER: E-GEOD-8448 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Optimization of experimental design parameters for high-throughput chromatin immunoprecipitation studies.

Ponzielli Romina R Boutros Paul C PC Katz Sigal S Stojanova Angelina A Hanley Adam P AP Khosravi Fereshteh F Bros Christina C Jurisica Igor I Penn Linda Z LZ

Nucleic acids research 20081021 21

High-throughput, microarray-based chromatin immunoprecipitation (ChIP-chip) technology allows in vivo elucidation of transcriptional networks. However this complex is not yet readily accessible, in part because its many parameters have not been systematically evaluated and optimized. We address this gap by systematically assessing experimental-design parameters including antibody purity, dye-bias, array-batch, inter-day hybridization bias, amplification method and choice of hybridization control ...[more]

PMID: 18940864

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Project description:We performed chromatin immunoprecipitation on microarray (ChIP-chip) transcriptional profiling of the SsrB regulator of Salmonella enterica Typhimurium to better characterize its regulon and to identify the DNA-recognition element coordinating its specific interaction at cis-regulatory sites. SsrB, the response regulator of the two component regulatory system SsrA-SsrB encoded within the Salmonella Pathogenicity Island (SPI-2), directs transcriptional activation of the closely associated type three secretion system (T3SS) also encoded at this locus. Immunoprecipitaiton of SsrB translationally fused to a C-terminal FLAG-tag from formaldehyde cross-linked genomic DNA was performed under SPI-2 activating and non-activating conditions. Downstream analysis and experimental work identified specific interactions within SPI-2 at the previously identified ssrA, ssaB, sseA, ssaG, ssaM promoters and identified an additional cryptic promoter driving expression upstream of ssaR. Additionally, a previously unknown SsrB interaction site within ssaE was shown to be the major contributor driving the expression of the downstream genes and not the previously identified interaction site within the intergenic region of ssaE-sseA. Interactions were also identified outside of SPI-2 upstream of other T3SS-associated genes encoded within other genomic islands, and for other uncharacterized genes. Integration of this data with transcriptional microarray work, previously published DNase I footprinting data, bacteria 1-hybrid investigations and comparative genomics analyses of cis-regulatory regions within the orthologous Sodalis symbiosis region 3 (SSR-3) of Sodalis glossinidius enabled identification of a conserved 18bp palindrome which was experimentally validated as being required for transcriptional activation of SsrB dependant genes. SsrB-FLAG immunoprecipitations were performed under SsrB activating and non-activating conditions from formaldehyde cross-linked bacterial lysates. Nine immunoprecipitation reactions were pooled into three replicate samples for the activating condition and three reactions were pooled into one sample for the non-activating control condition. The immunoprecipitated DNA in addition to the non-immunoprecipitated control DNA for each of the four samples were labeled with Cy3 or Cy5 fluorophores respectively and were concurrently hybridized to four arrays on a single slide.

Dataset Information

Characterization of technical variation in ChIP-on-chip studies

Publications

Optimization of experimental design parameters for high-throughput chromatin immunoprecipitation studies.

Similar Datasets

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