GSE74931-GSM1937963-GPL8708-PMID:26670385.tsv 6.42 KB
"genotype/variation: lacking the transcription factor Fur"	"characteristics_ch1.1"
"ip antibody: affinity purified anti-Fur antibody"	"characteristics_ch1.2"
"genotype/variation: lacking the transcription factor Fur"	"characteristics_ch2.1"
"sample type: input control"	"characteristics_ch2.2"
"NimbleScan software package, version 2.5 (Roche NimbleGen) was used to extract the scanned data, which was subsequently qunatile normalized using the statistical program R. The sample data table contains the log2 ratio of the IP/Input after data was quantile normalized."	"data_processing.1"
"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 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 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. 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."	"extract_protocol_ch1.1"
"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."	"extract_protocol_ch1.2"
"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 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 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. 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."	"extract_protocol_ch2.1"
"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."	"extract_protocol_ch2.2"
"Cultures grown in MOPS minimal glucose media containing 10 µM FeSO4"	"growth_protocol_ch1.1"
"Cultures grown in MOPS minimal glucose media containing 10 µM FeSO4"	"growth_protocol_ch2.1"
"Escherichia coli str. K-12 substr. MG1655"	"organism_ch1.1"
"Escherichia coli str. K-12 substr. MG1655"	"organism_ch2.1"
"IP ∆fur Anaerobic"	"source_name_ch1.1"
"Input ∆fur Anaerobic"	"source_name_ch2.1"
"∆fur Anaerobic [IP vs nput]"	"title.1"
"genotype/variation: lacking the transcription factor Fur"	"characteristics_ch1.1"
"ip antibody: affinity purified anti-Fur antibody"	"characteristics_ch1.2"
"genotype/variation: lacking the transcription factor Fur"	"characteristics_ch2.1"
"sample type: input control"	"characteristics_ch2.2"
"Cultures grown in MOPS minimal glucose media containing 10 µM FeSO4"	"growth_protocol_ch1.1"
"Cultures grown in MOPS minimal glucose media containing 10 µM FeSO4"	"growth_protocol_ch2.1"
"Escherichia coli str. K-12 substr. MG1655"	"organism_ch1.1"
"Escherichia coli str. K-12 substr. MG1655"	"organism_ch2.1"
"IP ∆fur Anaerobic"	"source_name_ch1.1"
"Input ∆fur Anaerobic"	"source_name_ch2.1"