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Sample GSM1550080 Query DataSets for GSM1550080
Status Public on Feb 02, 2015
Title AkgR_RSP_0981_3xMyc_ChIP_Seq
Sample type SRA
 
Source name Bacteria culture (SIS - Photo)
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)
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.
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
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2500
 
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
 
Submission date Nov 19, 2014
Last update date May 15, 2019
Contact name Timothy J Donohue
E-mail(s) tdonohue@bact.wisc.edu
Phone 608-265-8465
Organization name University of Wisconsin - Madison
Department Bacteriology
Lab Timothy Donohue
Street address 1552 University Avenue
City Madison
State/province WI
ZIP/Postal code 53726
Country USA
 
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
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 SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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