Cells grew in MOPS minimal medium with 0.4% glucose and 0.2 mM K2HPO4 and harvested at the OD600 value of 1.0.
Extracted molecule
genomic DNA
Extraction protocol
Cultures were grown to the OD600 value of 1.0 and treated with 1% formaldehyde for 10 min. To quench the reaction, glycine was added at the final concentration of 0.125 M for 5 min. Cells were washed twice with lysis buffer (10 mM Tris-HCl (pH 7.4), 0.1 M NaCl, 1 mM EDTA and 0.5% Tween-20). The washed cells were then lysed with the lysis buffer containing 8 KU/ml lysozyme, 1 mM PMSF and protease inhibitor cocktail (Sigma) for 30 min at 4 ℃. The lysates were sonicated by a sonicator (Bioruptor) and this sonication resulted in the size of DNA ranged from 100 bps to 1000 bps with the average size of 500 bps. After sonication, the lysates were centrifuged at 12,000 g for 20 min at 4 ℃ and the resulting supernatant was used for immunoprecipitation. Before immunoprecipitation, the magnetic beads coated with Dynabeads Protein G (Invitrogen) were pre-incubated with 0.05 mg/ml anti-FLAG antibody (Sigma) and the lysates were also pre-cleared by incubating with the beads only. To immunoprecipitate the PhoB-FLAG-DNA complex, beads pre-incubated with antibody were added in both lysates of MG1655_PhoB_FLAG and MG1655 strains at 4 ℃ overnight. The beads were washed once with IP buffer (10 mM Tris-HCl (pH 7.4), 0.1 M NaCl, 1 mM EDTA, and 0.05% [v/v] Tween-20 and 1 mM fresh PMSF), twice with ChIP wash buffer I (10 mM Tris HCl (pH 7.4), 300 mM NaCl, 1 mM EDTA, 0.1% Tween-20 and 1 mM fresh PMSF), three times with ChIP wash buffer II (10 mM Tris-HCl (pH 7.4), 500 mM NaCl, 1 mM EDTA, 0.1% [v/v] Tween-20 and 1 mM fresh PMSF), once with ChIP wash buffer III (10 mM Tris-HCl (pH 7.4), 250 mM LiCl, 1 mM EDTA, 0.1% [v/v] Tween-20 and 1 mM fresh PMSF) and once with TE buffer (10 mM Tris-HCl (pH 7.4) and 1 mM EDTA). After removing the TE buffer, beads were incubated with elution buffer (50 mM Tris-HCl (pH 7.4), 10 mM EDTA and 1% SDS) at 65 ℃ for 15 min twice and then the resulting eluted solutions combined. After incubating with the final concentration of 10.5 U/ml proteinase K (Sigma) at 42 ℃ for 2 hours, the reverse cross-link procedure was performed by incubating at 65 ℃ overnight. Samples were then treated with the final concentration of 26 μg/ml RNase A (Sigma). The PCR purification kit (Qiagen) was used to purify DNA from the RNase A-treated samples.
Label
Cy5
Label protocol
The instructions of the NimbleChipTM Arrays User Guide-ChIP-chip anaysis (version 2.0) for Cy5 and Cy3 labeling were followed.
Cells grew in MOPS minimal medium with 0.4% glucose and 0.2 mM K2HPO4 and harvested at the OD600 value of 1.0.
Extracted molecule
genomic DNA
Extraction protocol
Cultures were grown to the OD600 value of 1.0 and treated with 1% formaldehyde for 10 min. To quench the reaction, glycine was added at the final concentration of 0.125 M for 5 min. Cells were washed twice with lysis buffer (10 mM Tris-HCl (pH 7.4), 0.1 M NaCl, 1 mM EDTA and 0.5% Tween-20). The washed cells were then lysed with the lysis buffer containing 8 KU/ml lysozyme, 1 mM PMSF and protease inhibitor cocktail (Sigma) for 30 min at 4 ℃. The lysates were sonicated by a sonicator (Bioruptor) and this sonication resulted in the size of DNA ranged from 100 bps to 1000 bps with the average size of 500 bps. After sonication, the lysates were centrifuged at 12,000 g for 20 min at 4 ℃ and the resulting supernatant was used for immunoprecipitation. Before immunoprecipitation, the magnetic beads coated with Dynabeads Protein G (Invitrogen) were pre-incubated with 0.05 mg/ml anti-FLAG antibody (Sigma) and the lysates were also pre-cleared by incubating with the beads only. To immunoprecipitate the PhoB-FLAG-DNA complex, beads pre-incubated with antibody were added in both lysates of MG1655_PhoB_FLAG and MG1655 strains at 4 ℃ overnight. The beads were washed once with IP buffer (10 mM Tris-HCl (pH 7.4), 0.1 M NaCl, 1 mM EDTA, and 0.05% [v/v] Tween-20 and 1 mM fresh PMSF), twice with ChIP wash buffer I (10 mM Tris HCl (pH 7.4), 300 mM NaCl, 1 mM EDTA, 0.1% Tween-20 and 1 mM fresh PMSF), three times with ChIP wash buffer II (10 mM Tris-HCl (pH 7.4), 500 mM NaCl, 1 mM EDTA, 0.1% [v/v] Tween-20 and 1 mM fresh PMSF), once with ChIP wash buffer III (10 mM Tris-HCl (pH 7.4), 250 mM LiCl, 1 mM EDTA, 0.1% [v/v] Tween-20 and 1 mM fresh PMSF) and once with TE buffer (10 mM Tris-HCl (pH 7.4) and 1 mM EDTA). After removing the TE buffer, beads were incubated with elution buffer (50 mM Tris-HCl (pH 7.4), 10 mM EDTA and 1% SDS) at 65 ℃ for 15 min twice and then the resulting eluted solutions combined. After incubating with the final concentration of 10.5 U/ml proteinase K (Sigma) at 42 ℃ for 2 hours, the reverse cross-link procedure was performed by incubating at 65 ℃ overnight. Samples were then treated with the final concentration of 26 μg/ml RNase A (Sigma). The PCR purification kit (Qiagen) was used to purify DNA from the RNase A-treated samples.
Label
Cy3
Label protocol
The instructions of the NimbleChipTM Arrays User Guide-ChIP-chip anaysis (version 2.0) for Cy5 and Cy3 labeling were followed.
Hybridization protocol
The instructions of the NimbleChipTM Arrays User Guide-ChIP-chip anaysis (version 2.0) for hybridization were followed and the hybrization process was done by the NimbleGen hybridization system.
The R package, Ringo, was used to read the pair files and the limma package for within- and between-array normalization. ChIP-chip data were bi-weighted scaled within each array and aquantile normalized between arrays.
