genotype/variation: expressing the FLAG-tagged SigD in WT background growth phase: exponential phase
Growth protocol
C. glutamicum is grown in 100 ml of nutrient rich A medium containing 1% glucose in 500 ml flask at 33˚C
Extracted molecule
genomic DNA
Extraction protocol
C. glutamicum cultures at the late exponential phase (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-FLAG-tag antibody (Sigma). 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.
genotype/variation: expressing the FLAG-tagged SigD in WT background growth phase: exponential phase
Growth protocol
C. glutamicum is grown in 100 ml of nutrient rich A medium containing 1% glucose in 500 ml flask at 33˚C
Extracted molecule
genomic DNA
Extraction protocol
C. glutamicum cultures at the late exponential phase (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-FLAG-tag antibody (Sigma). 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
Hybridization was performed using Agilent Oligo aCGH Hybridization kit according to the manufacture’s manual (Agilent). Equal amount (2.5 μg) of each labeled DNA were mixed and hybridized to microarrays in an Agilent Technologies Microarray chamber at 65˚C for 24 h in a rotating Agilent hybridization oven at 20 rpm. After hybridization, microarrays were washed 5 min at room temperature with Oligo aCGH/ChIP-on-chip Wash Buffer 1 (Agilent), 5 min with 31˚C Oligo aCGH/ChIP-on-chip Wash buffer 2 (Agilent Technologies), 10 sec with acetonitrile, and 30 sec with Stabilization and Drying Solution (Agilent) at room temperature.
Scan protocol
Slides were scanned immediately after washing and placement within an Agilent Ozone Barrier Slide cover (Agilent) the Agilent DNA Microarray Scanner (G2505C) using two color scan setting for 4x44k array Scan resolution 5 microns. Scanning was done in both channels and PMT is set to 100% for both Cy3\cy5 channels.
Data processing
Feature extraction software provided by Agilent (version 10.5.1.1, Agilent Technologies, Palo Alto, CA, USA) was used to quantify the intensity of the fluorescent images and to normalize the results by subtracting local background fluorescence, according to the manufacturer’s instructions.
