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Status |
Public on Feb 02, 2015 |
Title |
AkgR_RSP_0981_3xMyc_ChIP_Seq |
Sample type |
SRA |
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Source name |
Bacteria culture (SIS - Photo)
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Organism |
Cereibacter sphaeroides |
Characteristics |
strain: ΔRSP_0981 complemented with 3xMyc tagged RSP_0981 from an IPTG inducible plasmid chip-antibody: Myc epitope antibody (ab9132, Abcam plc)
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Growth protocol |
R. sphaeroides strains that were grown in 500 ml cultures in roux bottles either photosynthetically (bubbling with 95% N2, 5% CO2) or aerobically (bubbling with 69% N2, 30% O2, 1% CO2) on Sistrom's minimal media and harvested at exponential phase. Where necessary 1 to 5μL of IPTG was added at the start of the culture to induce protein expression.
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Extracted molecule |
genomic DNA |
Extraction protocol |
At midexponential phase (~2×108 colony-forming units/ml), formaldehyde and sodium phosphate were added to a final concentration of 1% and 10 mM, respectively. This mixture was incubated at 30 °C for 4 min before glycine was added to 100 mM, and the solution was placed on ice for 30 min with gentle agitation to quench excess formaldehyde. Cells were centrifuged at 3000g, washed twice with chilled phosphate buffered saline, centrifuged, and flash-frozen at −80 °C. About 2×1010 cells were suspended in 0.5 ml of 100 mM Tris (pH 8.0), 300 mM NaCl, 2% Triton X-100, RNase A (0.5 μg) and 1 mM phenylmethylsulfonyl fluoride, and sonicated in a Misonix water bath for 8 mins with 10s pulses and intervals. Cell debris was removed by centrifugation for 10 min at 12,000g, and an aliquot was removed to analyze DNA fragmentation by agarose gel eletrophoresis (desired size of ~200–1000 bp with enrichment for ~500-bp molecules). The resulting supernatant was incubated with gentle mixing with 20 μl of Staphylococcus aureus protein A Sepharose beads (Sigma-Aldrich, St. Louis, MO) for 3 h at 4 °C as pretreatment to remove potential nonspecific binding to the beads. After the beads had been removed by centrifugation (5 min at 3000g), one-tenth of the sample was removed and used as non-antibody-treated control. Five microlitre of Myc epitope antibody (ab9132, Abcam plc) was added and the mixture was incubated overnight at 4 °C with gentle mixing before being incubated with protein A Sepharose beads (30 μl) for 3 h at 4 °C. The beads were recovered by centrifugation; washed once (4 °C) with 250 mM LiCl, 100 mM Tris (pH 8.0), and 2% Triton X-100; washed twice with 600 mM NaCl, 100 mM Tris (pH 8.0), and 2% Triton X-100; washed twice with 300 mM NaCl, 100 mM Tris (pH 8.0), and 2% Triton X-100; and washed twice with TE buffer (10 mM Tris pH 8.0 and 1 mM EDTA). Protein–DNA complexes were eluted from the beads by incubating at 65 °C for 30 min in 50 mM Tris (pH 8.0), 10 nM EDTA, and 1% sodium dodecyl sulfate. The beads were removed by centrifugation, and protein–DNA cross-linking was reversed by incubating the samples for 12 h at 65 °C. DNA was purified using the QIAquick PCR Purification Kit. Samples were sent to UW-Madison Biotech center for further processing and sequencing. Libraries were prepared according to Illumina's instructions by UW-Madison biotech center
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina HiSeq 2500 |
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Data processing |
Mapping was done to the R. sphaeroides reference genome using SOAP version 2.21 allowing for 2 mismatches for 50bp reads Peaks were identified using MOSAiCS at a false discovery rate of 0.05 using a two-sample analysis with Input DNA or mock chip used as control. Genome_build: ftp://ftp.ncbi.nih.gov/genomes/Bacteria/Rhodobacter_sphaeroides_2_4_1_uid57653/ Supplementary_files_format_and_content: Processed data files are provided as wig files with scores representing the normalized ratio of reads between a sample ChIP and INPUT DNA
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Submission date |
Nov 19, 2014 |
Last update date |
May 15, 2019 |
Contact name |
Timothy J Donohue |
E-mail(s) |
tdonohue@bact.wisc.edu
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Phone |
608-265-8465
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Organization name |
University of Wisconsin - Madison
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Department |
Bacteriology
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Lab |
Timothy Donohue
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Street address |
1552 University Avenue
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City |
Madison |
State/province |
WI |
ZIP/Postal code |
53726 |
Country |
USA |
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Platform ID |
GPL18841 |
Series (2) |
GSE63449 |
Genome-wide protein-DNA interaction analysis of CceR and AkgR transcription factors |
GSE63450 |
CceR and AkgR regulate of central carbon and energy metabolism in α-Proteobacteria |
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Relations |
BioSample |
SAMN03200245 |
SRA |
SRX763383 |
Supplementary file |
Size |
Download |
File type/resource |
GSM1550080_AkgR_RSP_0981.wig.gz |
13.5 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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