strain: CSH50 chip antibody: Anti-FLAG (sigma-aldrich cat no F3165)
Treatment protocol
The culture was divided into two 20-ml volumes and the cells collected by centrifugation. The supernatant was removed and the two pellets were subjected to different treatments. One was resuspended in 1 ml of pre-warmed 1X EG-minimal medium pH 7.2. The second was resuspended in 1 ml of pre-warmed 1X EG-minimal medium pH 4.5. Each suspension was added to a final volume of 20 ml 1X EG minimal medium of the same pH in a 250 ml flask. Cells were grown at 37°C and 200 rpm shaking for 90 min before harvesting for analysis.
Growth protocol
Strains were grown overnight in LB broth and then equalised to 0.15 optical density (OD600nm) units. Cells were collected and washed in EG minimal medium (pH 7.2). This wash step was repeated twice more with centrifugation to collect the cells. The final pellet was resuspended in 1 ml of 1X EG minimal (pH 7.2). Cells were grown to OD600 ~0.5-0.6 in a 40 ml volume in a 250 ml flask at 37°C and 200 rpm.
Extracted molecule
genomic DNA
Extraction protocol
OD600 15 units of cells were harvested for each ChIP procedure. Cells were harvested by centrifugation at 4,000 rpm for 8 min at room temperature and resuspended in 50 ml of pre-warmed PBS (37°C) in a 250 ml flask. DNA-protein and protein-protein interactions were cross-linked by adding 1,351 µl of formaldehyde drop-wise to a final concentration of 1%. Samples were cross-linked at room temperature with stirring for 30 min. Glycine was added to a final concentration of 0.125 M with stirring for 5 min at room temperature. Cells were centrifuged at 4,000 rpm for 8 min at 4°C and the supernatant removed. The pellet was re-suspended in 0.6 ml of lysis buffer containing 50 mM Tris-HCl, 10 mM EDTA, 1% SDS and incubated on ice for 10 min. 1.4 ml of IP dilution buffer (20 mM Tris-HCl pH 8.1, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.01% SDS, Roche protease inhibitor cocktail) was added and the chromatin was sonicated on ice in a 5ml tube to reduce the DNA length to an average size of approximately 500 bp using the Sanyo/MES Soniprep sonicator (8 bursts at an amplitude 10 microns for 30 seconds, with 1 minute cooling between bursts). This was transferred to a 2 ml microfuge tube and spun at 13,200 rpm for 10 min at 4°C. Supernatant was removed and 1 ml dilution buffer added.The chromatin material was precleared by adding 50 µl of normal rabbit immunoglobulin G (IgG, 1 mg/ml, Millipore). Samples were incubated for 1 h at 4°C on a rotating wheel. 100 µl of homogenous protein G-agarose (Roche) was added to the precleared chromatin and the samples were incubated for 3-5 h at 4°C on a rotating wheel. Samples were centrifuged at 4,000 rpm for 2 min at 4°C and this supernatant was used to set up immunoprecipitation reactions. 200 µl chromatin was removed and used as an input sample. Experimental immunoprecipitation were set up using 1,350 µl chromatin and 3.3 µg/µl of anti-FLAG antibody (Sigma). Mock immunoprecipitation 1,350 µl chromatin and 10 µg of normal mouse IgG (Millipore). Samples were incubated overnight on a rotating wheel at 4°C. Samples were centrifuged at 13,000 rpm for 5 min at 4°C and the supernatant was transferred to 2 ml microfuge tubes. 50 µl of protein G-agarose was added to each sample and incubated at 4°C for 3 h. The samples were centrifuged at 7,500 rpm for 2 min at 4°C to pellet the protein G-agarose beads. The supernatant was removed and the protein G-agarose beads were carefully washed. 91 For each wash, the wash buffer was added; the samples were vortexed briefly and were centrifuged at 7,500 rpm for 2 min at 4°C. The samples were left to stand on ice for 1 min before removing the supernatant. The washes were carried out in the following sequence: The beads were washed twice with 750 µl of cold IP wash buffer 1 containing 20 mM Tris-HCl (pH 8.1), 50 mM NaCl, 2mM EDTA, 1% Triton X-100, 0.1% SDS. The beads were transferred to a 1.5 ml microfuge tube after the first wash. The beads were washed once with 750 µl of cold IP wash buffer 2 containing 10 mM Tris-HCl (pH 8.1), 250 mM LiCl, 1 mM EDTA, 1% NP-40, 1% deoxycholic acid. The beads were washed twice with 750 µl of ice-cold TE buffer containing 10 mM Tris base (pH 8) and 1 mM EDTA. DNA-protein-antibody complexes were eluted from the protein G-agarose beads by adding 225 µl of IP elution buffer containing 100 mM NaHCO3 and 1% SDS. The beads were resuspended in IP elution buffer, briefly vortexed and centrifuged at 7,500 rpm for 2 min at room temperature. The supernatant was collected in fresh 1.5 ml microfuge tubes. The bead pellets in the original tubes were resuspended in 225 µl of IP elution buffer again and briefly vortexed and centrifuged at 7,500 rpm for 2 min. IP and input samples were thawed on ice. 0.1 µl of RNase A (10 mg/ml, ICN Biochemicals) and 16 µl of 5 M NaCl (to a final concentration of 0.3 M) was added to the input sample. Similarly, 0.2 µl of RNase A and 27 µl of 5 M NaCl (to a final concentration of 0.3 M) was added to each IP sample. All samples including the input DNA were incubated at 65°C for 6 h to reverse cross-links. 9 µl of Proteinase K (10 mg/ml, GibcoBRL) was added to each sample and incubated overnight at 45°C. 2 µl of yeast tRNA (5mg/ml, Invitrogen) was added to each sample followed by 250 µl of phenol (pH 8, Sigma) and 250 µl of chloroform. Samples were centrifuged at 13,000 rpm for 5 min at room temperature. The aqueous layer was collected in fresh 1.5 ml centrifuge tubes and 500 µl of chloroform was added. The samples were vortexed and centrifuged at 13,000 rpm for 5 min and room temperature. The aqueous layer was transferred to a 2 ml microfuge tube. 5 µg of glycogen (5 mg/ml, Roche), 1 µl of tRNA (5 mg/ml, Invitrogen) and 50 µl of 3 M NaAc (pH 5.2) was added to each sample and mixed well. The DNA was precipitated with 1,375 µl of 95% ethanol and incubated at -80°C for 1 h. The samples were centrifuged at 13,000 rpm for 20 min at 4°C. The DNA pellets were washed with 500 µl of ice-cold 70% ethanol and centrifuged at 13,000 rpm for 5 min at 4°C. The supernatant was removed and the samples were air-dried for 10-15 min. DNA pellets of IP samples were resuspended in 50 µl nuclease-free H2O. Input DNA samples were resuspended in 100 µl of nuclease-free H2O. Samples were incubated at 37°C for 1 h.
Label
Cy3
Label protocol
The BioPrime Array CGH kit (Invitrogen) was used to fluorescently label DNA for ChIP microarray experiments. 100 µl volume IP labelling reactions were set up as follows: 40 µl 2.5X random primers, 40 µl of IP DNA, 6.5 µl nuclease-free H2O, this was heated to 100°C for 10 min and chilled on ice for 5 min. To this sample, 10 µl of 10X dNTP mix (1mM dCTP, 2mM dGTP, 2mM dTTP, 2 mM dATP), 1.5 µl of Cy3/Cy5-dCTP (23 nmol GE Healthcare) and 2 µl exo-Klenow (40 U/µl) was added. Reactions were carried out at 37°C overnight in the dark. 15 µl of stop buffer was added to each sample to terminate the reaction. 200 ng of input DNA was labelled as described above. Samples were purified using Qiagen MinElute PCR purification kit as per manufacturer’s instructions.
The culture was divided into two 20-ml volumes and the cells collected by centrifugation. The supernatant was removed and the two pellets were subjected to different treatments. One was resuspended in 1 ml of pre-warmed 1X EG-minimal medium pH 7.2. The second was resuspended in 1 ml of pre-warmed 1X EG-minimal medium pH 4.5. Each suspension was added to a final volume of 20 ml 1X EG minimal medium of the same pH in a 250 ml flask. Cells were grown at 37°C and 200 rpm shaking for 90 min before harvesting for analysis.
Growth protocol
Strains were grown overnight in LB broth and then equalised to 0.15 optical density (OD600nm) units. Cells were collected and washed in EG minimal medium (pH 7.2). This wash step was repeated twice more with centrifugation to collect the cells. The final pellet was resuspended in 1 ml of 1X EG minimal (pH 7.2). Cells were grown to OD600 ~0.5-0.6 in a 40 ml volume in a 250 ml flask at 37°C and 200 rpm.
Extracted molecule
genomic DNA
Extraction protocol
OD600 15 units of cells were harvested for each ChIP procedure. Cells were harvested by centrifugation at 4,000 rpm for 8 min at room temperature and resuspended in 50 ml of pre-warmed PBS (37°C) in a 250 ml flask. DNA-protein and protein-protein interactions were cross-linked by adding 1,351 µl of formaldehyde drop-wise to a final concentration of 1%. Samples were cross-linked at room temperature with stirring for 30 min. Glycine was added to a final concentration of 0.125 M with stirring for 5 min at room temperature. Cells were centrifuged at 4,000 rpm for 8 min at 4°C and the supernatant removed. The pellet was re-suspended in 0.6 ml of lysis buffer containing 50 mM Tris-HCl, 10 mM EDTA, 1% SDS and incubated on ice for 10 min. 1.4 ml of IP dilution buffer (20 mM Tris-HCl pH 8.1, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.01% SDS, Roche protease inhibitor cocktail) was added and the chromatin was sonicated on ice in a 5ml tube to reduce the DNA length to an average size of approximately 500 bp using the Sanyo/MES Soniprep sonicator (8 bursts at an amplitude 10 microns for 30 seconds, with 1 minute cooling between bursts). This was transferred to a 2 ml microfuge tube and spun at 13,200 rpm for 10 min at 4°C. Supernatant was removed and 1 ml dilution buffer added.The chromatin material was precleared by adding 50 µl of normal rabbit immunoglobulin G (IgG, 1 mg/ml, Millipore). Samples were incubated for 1 h at 4°C on a rotating wheel. 100 µl of homogenous protein G-agarose (Roche) was added to the precleared chromatin and the samples were incubated for 3-5 h at 4°C on a rotating wheel. Samples were centrifuged at 4,000 rpm for 2 min at 4°C and this supernatant was used to set up immunoprecipitation reactions. 200 µl chromatin was removed and used as an input sample. Experimental immunoprecipitation were set up using 1,350 µl chromatin and 3.3 µg/µl of anti-FLAG antibody (Sigma). Mock immunoprecipitation 1,350 µl chromatin and 10 µg of normal mouse IgG (Millipore). Samples were incubated overnight on a rotating wheel at 4°C. Samples were centrifuged at 13,000 rpm for 5 min at 4°C and the supernatant was transferred to 2 ml microfuge tubes. 50 µl of protein G-agarose was added to each sample and incubated at 4°C for 3 h. The samples were centrifuged at 7,500 rpm for 2 min at 4°C to pellet the protein G-agarose beads. The supernatant was removed and the protein G-agarose beads were carefully washed. 91 For each wash, the wash buffer was added; the samples were vortexed briefly and were centrifuged at 7,500 rpm for 2 min at 4°C. The samples were left to stand on ice for 1 min before removing the supernatant. The washes were carried out in the following sequence: The beads were washed twice with 750 µl of cold IP wash buffer 1 containing 20 mM Tris-HCl (pH 8.1), 50 mM NaCl, 2mM EDTA, 1% Triton X-100, 0.1% SDS. The beads were transferred to a 1.5 ml microfuge tube after the first wash. The beads were washed once with 750 µl of cold IP wash buffer 2 containing 10 mM Tris-HCl (pH 8.1), 250 mM LiCl, 1 mM EDTA, 1% NP-40, 1% deoxycholic acid. The beads were washed twice with 750 µl of ice-cold TE buffer containing 10 mM Tris base (pH 8) and 1 mM EDTA. DNA-protein-antibody complexes were eluted from the protein G-agarose beads by adding 225 µl of IP elution buffer containing 100 mM NaHCO3 and 1% SDS. The beads were resuspended in IP elution buffer, briefly vortexed and centrifuged at 7,500 rpm for 2 min at room temperature. The supernatant was collected in fresh 1.5 ml microfuge tubes. The bead pellets in the original tubes were resuspended in 225 µl of IP elution buffer again and briefly vortexed and centrifuged at 7,500 rpm for 2 min. IP and input samples were thawed on ice. 0.1 µl of RNase A (10 mg/ml, ICN Biochemicals) and 16 µl of 5 M NaCl (to a final concentration of 0.3 M) was added to the input sample. Similarly, 0.2 µl of RNase A and 27 µl of 5 M NaCl (to a final concentration of 0.3 M) was added to each IP sample. All samples including the input DNA were incubated at 65°C for 6 h to reverse cross-links. 9 µl of Proteinase K (10 mg/ml, GibcoBRL) was added to each sample and incubated overnight at 45°C. 2 µl of yeast tRNA (5mg/ml, Invitrogen) was added to each sample followed by 250 µl of phenol (pH 8, Sigma) and 250 µl of chloroform. Samples were centrifuged at 13,000 rpm for 5 min at room temperature. The aqueous layer was collected in fresh 1.5 ml centrifuge tubes and 500 µl of chloroform was added. The samples were vortexed and centrifuged at 13,000 rpm for 5 min and room temperature. The aqueous layer was transferred to a 2 ml microfuge tube. 5 µg of glycogen (5 mg/ml, Roche), 1 µl of tRNA (5 mg/ml, Invitrogen) and 50 µl of 3 M NaAc (pH 5.2) was added to each sample and mixed well. The DNA was precipitated with 1,375 µl of 95% ethanol and incubated at -80°C for 1 h. The samples were centrifuged at 13,000 rpm for 20 min at 4°C. The DNA pellets were washed with 500 µl of ice-cold 70% ethanol and centrifuged at 13,000 rpm for 5 min at 4°C. The supernatant was removed and the samples were air-dried for 10-15 min. DNA pellets of IP samples were resuspended in 50 µl nuclease-free H2O. Input DNA samples were resuspended in 100 µl of nuclease-free H2O. Samples were incubated at 37°C for 1 h.
Label
Cy5
Label protocol
The BioPrime Array CGH kit (Invitrogen) was used to fluorescently label DNA for ChIP microarray experiments. 100 µl volume IP labelling reactions were set up as follows: 40 µl 2.5X random primers, 40 µl of IP DNA, 6.5 µl nuclease-free H2O, this was heated to 100°C for 10 min and chilled on ice for 5 min. To this sample, 10 µl of 10X dNTP mix (1mM dCTP, 2mM dGTP, 2mM dTTP, 2 mM dATP), 1.5 µl of Cy3/Cy5-dCTP (23 nmol GE Healthcare) and 2 µl exo-Klenow (40 U/µl) was added. Reactions were carried out at 37°C overnight in the dark. 15 µl of stop buffer was added to each sample to terminate the reaction. 200 ng of input DNA was labelled as described above. Samples were purified using Qiagen MinElute PCR purification kit as per manufacturer’s instructions.
Hybridization protocol
Samples were prepared for hybridisation as follows: 50 µl 2X Hi-RPM Hybridisation buffer (Agilent), 25 µl ChIP DNA, 5 µl input DNA, 10 µl 10X GE blocking Agent (Agilent) and 10 µl nuclease-free H2O. Samples were added to a microfuge tube, vortexed briefly and collected by short centrifugation spin. 100 µl was added to an array on the microarray slides. The arrays were sealed with the appropriate gasket slide and loaded into the Agilent Microarray Hybridisation Chamber Kit (G2534A). Microarrays were hybridised for 24 h at 65°C in a hybridization oven (Agilent). Slides were washed in wash buffer 1 followed by wash buffer 2 as described below. 200 ml of buffer was used for each wash step. Wash buffers were made up as 1 L solutions and filter sterilised using 0.22 µM pore membrane (Nalgene) as follows: Wash buffer 1: 1.4 L ddH2O, 0.6 L 20X SSC (Sigma), 1 ml 10% Triton. Wash buffer 2: 994.5 L ddH2O, 5 ml 20X SSC (Sigma), 500 µl 10% Triton. The slide was removed from the hybridisation oven and prised apart whilst submerged in wash buffer 1 that was pre-warmed to 60°C. The slide was then washed in buffer 1 at 37°C for 10 min with stirring, followed by washing in wash buffer 1 at room temperature for 10 min. The slide was then washed in wash buffer 2 at room temperature for 5 min with stirring; this was followed by a second wash in fresh wash buffer 2 for 5 min.
Scan protocol
Slides were scanned on an Agilent G2565CA Microarray Scanner
Data processing
Agilent Feature Extraction Software version 10.5.1.1.
ChIP-on-chip of OmpR binding in Salmonella enterica serovar Typhimurium strain SL1344 and E.scherichia coli strain CSH50 at pH 7 and pH 4.5 including analysis of OmpR binding in SL1344 in the absence and presence of novobiocin (25 μg/ml)
