genotype: glxR::Strep-tag II, ΔcyaB strain: KT25 antibody: anti-Strep-tag II antibody vendor: Qiagen antibody catalog #: 34850 antibody lot #: 130170795
Treatment protocol
Formaldehyde was added to cultures at a final concentration of 1% (v/v) and incubation was continued for 20 min
Growth protocol
C. glutamicum was grown in A medium containing 1% glucose at 33oC under aeration for 240min to reach the late-exponential phase of growth.
Extracted molecule
genomic DNA
Extraction protocol
Exponentially-growing C. glutamicum cultures (35 ml) at the OD610 of up to 2.5 were treated with formaldehyde (at the final concentration of 1%) and incubate for 20 min at room temperature. The cross-linking was quenched by addition of glycine (at the final concentration of 125 mM) and incubation for 10 min at room temperature. Cells were then collected by centrifugation, washed twice with Tris-buffered saline (20 mM Tris-HCl pH 7.5, 150 mM NaCl) and stored at -80˚C. Pellets were resuspended in 2 ml IP buffer (50 mM HEPES-KOH pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, Roche Antiprotease mini). The cells were mechanically disrupted for nine cycles of 25 s at a speed rating of 6.5 with a FastPrep™ FP120 (BIO 101, Thermo Savant) at room temperature with intermittent cooling on ice for 5 min. The supernatant after centrifugation was sonicated on ice to shear DNA to an average size to 600-1,000 bp. A 50 μl fraction of the supernatant was saved for later analysis (reference DNA). The remainder was subjected to immunoprecipitation with 100μl of magnetic beads coated with Protein G (Invitrogen), which was coupled to the monoclonal anti-Strep-tag II antibody (Qiagen). The mixture was incubated overnight on a rotating platform at 4˚C. The beads were washed once with IP buffer, twice with IP buffer containing 400 mM NaCl, eight times with RIPA buffer (50 mM HEPES pH7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Sodium deoxycholate), and twice with TE buffer with 50 mM NaCl. Immunoprecipitated complexes were eluted from the beads by treatment with 210 μl elution buffer (50 mM Tris-HCl pH8.0, 10 mM EDTA, 1% SDS) at 65˚C for 20 min. Cross-links of immunoprecipitated samples and of total DNA samples were reversed by incubation overnight at 65˚C. Samples were then treated with RNase A and proteinase K for 2 h at 55˚C. DNA was extracted with phenol-chloroform and purified with QIAquick PCR purification mini Elute kit (Qiagen).
Label
Cy5
Label protocol
DNA samples were blunted with T4 DNA polymerase, ligated to linkers, and amplified by PCR. Amplified DNA from reference DNA and immunoprecipitated DNA were differentially labeled with Cy3 and Cy5, respectively, by using CGH labeling kit (Invitrogen) according to the manufacture’s instruction.
Formaldehyde was added to cultures at a final concentration of 1% (v/v) and incubation was continued for 20 min
Growth protocol
C. glutamicum was grown in A medium containing 1% glucose at 33oC under aeration for 240min to reach the late-exponential phase of growth.
Extracted molecule
genomic DNA
Extraction protocol
Exponentially-growing C. glutamicum cultures (35 ml) at the OD610 of up to 2.5 were treated with formaldehyde (at the final concentration of 1%) and incubate for 20 min at room temperature. The cross-linking was quenched by addition of glycine (at the final concentration of 125 mM) and incubation for 10 min at room temperature. Cells were then collected by centrifugation, washed twice with Tris-buffered saline (20 mM Tris-HCl pH 7.5, 150 mM NaCl) and stored at -80˚C. Pellets were resuspended in 2 ml IP buffer (50 mM HEPES-KOH pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, Roche Antiprotease mini). The cells were mechanically disrupted for nine cycles of 25 s at a speed rating of 6.5 with a FastPrep™ FP120 (BIO 101, Thermo Savant) at room temperature with intermittent cooling on ice for 5 min. The supernatant after centrifugation was sonicated on ice to shear DNA to an average size to 600-1,000 bp. A 50 μl fraction of the supernatant was saved for later analysis (reference DNA). The remainder was subjected to immunoprecipitation with 100μl of magnetic beads coated with Protein G (Invitrogen), which was coupled to the monoclonal anti-Strep-tag II antibody (Qiagen). The mixture was incubated overnight on a rotating platform at 4˚C. The beads were washed once with IP buffer, twice with IP buffer containing 400 mM NaCl, eight times with RIPA buffer (50 mM HEPES pH7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Sodium deoxycholate), and twice with TE buffer with 50 mM NaCl. Immunoprecipitated complexes were eluted from the beads by treatment with 210 μl elution buffer (50 mM Tris-HCl pH8.0, 10 mM EDTA, 1% SDS) at 65˚C for 20 min. Cross-links of immunoprecipitated samples and of total DNA samples were reversed by incubation overnight at 65˚C. Samples were then treated with RNase A and proteinase K for 2 h at 55˚C. DNA was extracted with phenol-chloroform and purified with QIAquick PCR purification mini Elute kit (Qiagen).
Label
Cy3
Label protocol
DNA samples were blunted with T4 DNA polymerase, ligated to linkers, and amplified by PCR. Amplified DNA from reference DNA and immunoprecipitated DNA were differentially labeled with Cy3 and Cy5, respectively, by using CGH labeling kit (Invitrogen) according to the manufacture’s instruction.
Hybridization protocol
Fifty microlitres of each Cy3- and Cy5-labelled DNA was mixed and added to 100 µl 2x hybridization buffer [12x saline sodium citrate (SSC), 0.4 % SDS, 10x Denhardt's solution, 0.2 mg ml–1 denatured salmon sperm DNA]. All steps, from hybridizing to washing and drying, were automatically performed using a Lucidea Automated Slide Processor (Amersham Biosciences). The hybridization solution, which was heated at 95 °C for 2 min and cooled to room temperature, was added to hybridization buffer to a final combined volume of 200 µl. The DNA microarray was inserted into the Lucidea Automated Slide Processor and hybridization at 60 °C with the whole-genome DNA microarray was allowed to proceed for 14 h. The hybridization arrays were subsequently washed (once at 35 °C for 6 min in 2x SSC and 0.2 % SDS, once at 35 °C for 6 min in 0.2x SSC and 0.2 % SDS), rinsed (twice at 35 °C in 0.2x SSC and once at 37 °C in isopropanol) and dried.
Scan protocol
The slides were scanned at 532 nm for Cy3 and at 635 nm for Cy5 with a Fujifilm Fluorescent Image Analyser FLA-8000 (Fuji).
Data processing
Images were analysed with a GenePix Pro 5.0 instrument (Axon Instruments). Individual spots were located, the Cy3 and Cy5 fluorescence intensity at each spot was measured, and the background signal intensities were determined. Data was normalized with a GenePix Pro 5.0. Each data was normalized so that the mean of the ratio of means of all of the features, except the flagged ones, is equal to 1.
