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| Status |
Public on Dec 01, 2015 |
| Title |
Fur IP ChIP-Seq Aerobic B |
| Sample type |
SRA |
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| Source name |
Aerobic cultures
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| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
genotype/variation: Wild-type
growth medium: MOPS minimal glucose media containing 10 µM FeSO4
chip antibody: Custom anti-Fur polyclonal antibody
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| Treatment protocol |
Sodium phosphate (1/100 vol. of 1M, pH 7.6; 10 mM final) was added to the mid-log cultures followed by formaldehyde to 1% final, and aerobic or anaerobic sparging was continued for 10 min. Cold 2.5 M glycine was added to 100mM and the mixture was incubated at 4 °C with aerobic or anaerobic sparging for 30 minutes to stop the crosslinking. Cells were spun at 3500 x g, and washed repeatedly with phosphate buffered saline before being frozen at -80 °C.
Custom anti-Fur antibodies were purified over a His6-Fur bound HiTrap NHS-activated HP column (GE Healthcare) as previously described (PMID: 21478858). Western blot analyses showed that the purified antibody was specific for Fur.
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| Growth protocol |
Cells were grown aerobically (70% N2, 25% O2, and 5% CO2) or anaerobically (95% N2 and 5% CO2) until mid-log phase (OD600 of ~0.3-0.35).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cell pellets (from initial 250 mL of culture) were thawed and resuspended in 250 μL of IP buffer (100 mM Tris pH 8, 300 mM NaCl, 1% TritonX-100) and sonicated using a microtip sonicator set at 10% output for 20 second intervals with periods of cooling in between. Cells were then treated for one hour at 4 °C with RNase A (2 ng/ml), micrococcal nuclease (50 units), 20 μM CaCl2,1.2 mM KCl, 0.3 mM NaCl, 6 mM sucrose, and 10 μM DTT. EDTA was added to 10 mM to stop the micrococcal nuclease and the samples were spun down to remove cell debris. The lysate was then precleared through incubation with a 50/50 slurry of sepharose protein A beads in IP buffer for 2-3 hours at 4 °C. The beads were removed by centrifugation and antibody was added to the pre-cleared lysate for an overnight incubation. The next day, 30 μl of a 50/50 slurry of sepharose protein A beads in IP buffer was added to the lysate to capture antibody-protein-DNA complex for one hour at 4 °C. Beads were then washed once with 1 ml of LiCl wash buffer (100 mM Tris pH 8, 250 mM LiCl, 2% TritonX-100), twice with 600 mM NaCl wash buffer (100 mM Tris pH 8, 600 mM NaCl, 2% TritonX-100), twice with 300 mM NaCl wash buffer (100 mM Tris pH 8, 300 mM NaCl, 2% TritonX-100), and twice with TE. Elution buffer (50 mM Tris pH 8, 10 mM EDTA, 1% SDS) was added after the final wash step, and beads were incubated at 65 °C for 30 minutes to remove the crosslinked protein-DNA complexes from the beads. After centrifugation to remove the beads, the samples were incubated overnight at 65 °C to reverse the protein-DNA formaldehyde crosslinks. DNA was purified using Qiagen’s PCR Purification kit and eluted to a final volume of 50 μl with EB.
DNA samples were submitted to the University of Wisconsin-Madison DNA Sequencing Facility for ChIP-seq library preparation. All libraries were generated using reagents from the Illumina Paired End Sample Preparation Kit (Illumina) and the Illumina protocol “Preparing Samples for ChIP Sequencing of DNA” (Illumina part # 11257047 RevA) as per the manufacturer’s instructions, except products of the ligation reaction were purified by gel electrophoresis using 2% SizeSelect agarose gels (Invitrogen) targeting 50 bp fragments. After library construction and amplification, quality and quantity were assessed using an Agilent DNA 1000 series chip assay (Agilent) and QuantIT PicoGreen dsDNA Kit (Invitrogen), respectively, and libraries were standardized to 10μM. Cluster generation was performed using a cBot Single Read Cluster Generation Kit (v4) and placed on the Illumina cBot. For aerobic and anaerobic replicates A, a single read, 50 bp run was performed, using standard SBS kits (v4) and SCS 2.6 on an Illumina Genome Analyzer IIx. For aerobic and anaerobic replicates B and C, the input, and the anaerobic, iron deficient samples, a single read, 50 bp run was performed, using standard SBS kits (v3) and SCS 1.8.2 on an Illumina HiSeq2000.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Reformat Illumina files to Sanger format, FASTQ Groomer
Map reads to theEscherichia coli MG1655 K-12 genome, Bowtie 2
Call peaks to and FDR 0.1, MOSAiCs
Deconvolute peaks in close proximity, dPeak
Require peaks to be present in at least 2 replicates and conform to a peak shape by visual inspection
Scale data set to 20 million reads
Genome_build: Escherichia coli MG1655 K-12 genome version U00096.2
Supplementary_files_format_and_content: Processed data files contain the number of reads mapped to each base pair after the total number of reads were scaled to 20 million reads per experiment
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| Submission date |
Nov 12, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Patricia J Kiley |
| E-mail(s) |
pjkiley@wisc.edu
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| Organization name |
University of Wisconsin - Madison
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| Department |
Biomolecular Chemistry
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| Street address |
420 Henry Mall, Room 1135 Biochemistry Building
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| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53706 |
| Country |
USA |
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| Platform ID |
GPL15010 |
| Series (2) |
| GSE74932 |
The impact of anaerobiosis on expression of the iron-responsive Fur and RyhB Regulons [ChIP-seq] |
| GSE74933 |
The impact of anaerobiosis on expression of the iron-responsive Fur and RyhB Regulons |
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| Relations |
| BioSample |
SAMN04261715 |
| SRA |
SRX1427676 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1937965_Fur_IP_ChIP_seq_Aerobic_B_WIG.wig.gz |
165.7 Kb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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