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| Status |
Public on Dec 15, 2014 |
| Title |
PrrA_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: PrrA3 complemented with 3xMyc tagged PrrA from an IPTG inducible plasmid
chip-antibody: Myc epitope antibody
antibody manufacturer: Abcam plc
antibody catalog number: ab9132
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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) 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) or FnrL polyclonal antibody 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 2000 |
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| Data processing |
100 bp reads were trimmed to 70bp to remove unreliable base calls. 50bp reads were not trimmed
Mapping was done to the R. sphaeroides reference genome using SOAP version 2.21 allowing for 3 mismatches for 70bp reads and 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: Rhodobacter sphaeroides 2.4.1 GCA_000012905.2
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 |
Jun 20, 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 |
GPL18840 |
| Series (2) |
| GSE58716 |
Global analysis of photosynthesis transcriptional regulatory networks [ChIP-seq] |
| GSE58717 |
Global analysis of photosynthesis transcriptional regulatory networks |
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| Relations |
| BioSample |
SAMN02870012 |
| SRA |
SRX620396 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1417292_PrrA.wig.gz |
12.8 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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