|
| Status |
Public on Mar 10, 2020 |
| Title |
CFT073 WT CHIP-seq INPUT Replicate 1 |
| Sample type |
SRA |
| |
|
| Source name |
Bacterial Culture
|
| Organism |
Escherichia coli CFT073 |
| Characteristics |
media: MOPS minimal media (pH 7.4) 0.2% glucose
od600: 0.25
oxygen: Anaerobic
tissue: Bacterial Culture
|
| Treatment protocol |
. Cellular DNA was crosslinked to proteins by addition of 1% formaldehyde
|
| Growth protocol |
CFT073 wild-type (WAM 4505) strain replicate 1 was grown in a MOPS minimal medium (pH 7.4) supplemented with 0.2% glucose, which contains 10.0 μM FeSO4, an amount considered iron sufficient. Anaerobic growthof cultures was achieved by sparging cells with a gas mix containing 95% N2 and 5% CO2. Optical density (OD600nm) was measured in a Perkin Elmer Lambda 25 UV/Vis spectrophotometer.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were harvested at 7500 rpm for 10 min at 4°C followed by sonication using a water bath sonicator (Misonix) to shear the DNA to a fragment size of 150-400bp to facilitate library production. The cell lysate was incubated with antibody specific to Fur that was affinity purified over His6-Fur bound HiTrap N-hydroxy-succinimide (NHS)-activated high performance (HP) column (GE Healthcare) (72). DNA was reversed crosslinked and eluted from the DNA-protein complex using 50mM Tris pH 8.0, 10mM EDTA, 1% SDS.
Ten ng of purified input was used to construct DNA libraries as described in the Illumina TruSeq ChIP preparation kit (IP-202-1012), except the products of ligation reactions were purified using 2% size select agarose gel (Invitrogen). After library construction and amplification, quantity and quality were validated using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and Agilent 2100 Bioanalyzer before submitting them to University of Wisconsin-Madison DNA Sequencing facility for Illumina sequencing (Illumina HiSeq 2500) for single end 1X50bp reads, as per manufacturer recommendations.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
WT Replicate 1 Input DNA isolated from total cultures grown to OD600 of 0.25 after formaldehyde crosslinking.
|
| Data processing |
For ChIP-seq analysis. ChIP-seq sequence reads of low quality were removed from each file using Trimmomatic (version 0.30) using standard settings. Furthermore, the first and last five bases from each read were trimmed to remove the locations with the lowest average quality scores. This removed between 0.1% and 2.6% of reads. Filtered sequence reads were aligned to the published E. coli CFT073 genome (NC_004431.1) using the software package Bowtie 2 (version 2.2.2), using default settings. Sequence reads that didn’t align to the CFT073 genome at all or were aligned to more than one location were eliminated (an average of 15% of reads were removed). Areas of Fur enrichment were identified using MOSAiCS (version 1.6.0) with peaks with an FDR < 0.05 being included as significant. For visualization, aligned BAM files were converted into WIG files using QuEST (version 1.2) using standard settings. All WIG files were visualized in MochiView. For identification of Fur ChIP-seq peaks in the promoters of genes, a cut-off of 19.5 was selected for peak summit height.
Genome_build: NC_004431.1
Supplementary_files_format_and_content: RNA-seq processed data are RPKM normalized read counts. ChIP-seq processed data are in WIG format.
|
| |
|
| Submission date |
Feb 18, 2020 |
| Last update date |
Mar 10, 2020 |
| Contact name |
Kevin S Myers |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Wisconsin - Madison
|
| Department |
Great Lakes Bioenergy Research Center
|
| Street address |
5127 WEI, 1552 University Ave
|
| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53726 |
| Country |
USA |
| |
|
| Platform ID |
GPL18955 |
| Series (1) |
| GSE145424 |
Tailoring a global iron regulon to a uropathogen |
|
| Relations |
| BioSample |
SAMN14124212 |
| SRA |
SRX7737572 |
