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Analysis of ChIP-Seq data identifies BfmR regulon

ABSTRACT: Purpose:To identify the direct targets and regulatory mechanism of BfmR in Pseudomonas aeruginosa, we performed chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (Seq). Methods: In the experiment, chromatin bound BfmR was crosslinked, sheared, bound to antibody, nonspecific proteins and nucleic acids removed, purified and sequenced. The sequencing data were obtained from several independent ChIP samples and then located on the p. aeruginosa genome. The MACS (Model-based Analysis for ChIP-Seq) software was used to call peak for sequence reads. Results: We identified 141 enriched loci (p-value≤1e-5; fold enrichment>3) containing BfmR binding peak in the P. aeruginosa genome. In accordance with the previous studies, bfmR, PA4103 promoters were significantly enriched compared to input DNA (49, 60-fold, respectively), had the highest degree of enrichment in all peaks.

ORGANISM(S): Pseudomonas aeruginosa

PROVIDER: GSE154264 | GEO | 2021/04/13

REPOSITORIES: GEO

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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.

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Analysis of ChIP-Seq data identifies BfmR regulon

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