Citrobacter Rodentium HppXCROD2: A plasmid-encoded H-NS selectively binds its own plasmid
Citrobacter Rodentium HppXCROD2: A plasmid-encoded H-NS selectively binds its own plasmid
Cultures were crosslinked by adding formaldehyde to a final concentration of 1%, incubated for 20 minutes at room temperature, and quenched by adding glycine to a final concentration of 0.5 M.  Cells were washed once in original culture volume of 1X TBS then washed again in 1 mL of 1X TBS.  The supernatant was removed and the pellet was stored at -20 degrees C. 15 mL cultures grown in DMEM to an OD600 of ~0.8 (6 to 7h). Extraction and Library construction occur in the same protocol: Pelleted cells were resuspended in FA lysis buffer containing 2 mg/mL of lysozyme.  Samples were incubated for 30 minutes at 37 degrees C, chilled on ice for at least 5 minutes, and then sonicated for 30 minutes, with 30 s pulses followed by 30 s off.  Samples were spun at maximum speed in a microcentrifuge, the supernatants were removed and diluted to 1 mL lysate per 20 mL original culture volume.  Samples were immunoprecipitated according to the following procedure:  for every 800 uL of chromatin, 25 uL of protein A beads (50% TBS slurry), and 2 uL anti-FLAG antibody (M2) OR 2ul anti-s70 antibody were added.  Samples were incubated on rotisserie rotater for 90 minutes at room temperature.  Samples were then transferred to Spin-X columns (Corning) and washed twice with FA lysis buffer (150 mM NaCl), 3 minutes each wash, rotating at room temperature.  The samples were spun at 4000 rpm after each wash.  The samples were then washed twice in the same way with 10 mM Tris-HCl, pH 7.5. The immunoprecipitated DNA, still attached to protein A beads, were then treated with Quick Blunting Kit (NEB):  The samples were resuspended in the following mixture: 10 ul 10x quick blunting buffer, 10 ul dNTP mix (kit supplied), 80 ul dH2O, 2ul blunt enzyme mix.  The samples were incubated at room temperature, while rotating, for 30 minutes.  The samples were then washed twice with FA lysis buffer (150 mM NaCl), as described above, and then washed twice with 10 mM Tris-HCl, pH 8.0.  The samples were then A tailed, using Klenow (3'->5'exo-), from NEB:  The samples were resuspended in the following mixture: 10 ul 10X NEBuffer 2, 2 ul 100mM dATP (NEB), 88 ul dH2O, 2 ul Klenow.  The samples were incubated at 37 degrees, while rotating, for 30 minutes. The samples were then washed twice with FA lysis buffer (150 mM NaCl), as described above, and then washed twice with 10 mM Tris-HCl, pH 7.5.  Illumina adapters were then ligated to the samples using the NEB Quick Ligation kit.  The samples were resuspended in the following mixture:  100 ul 1x ligase buffer (50 ul 2x buffer + 50 ul dH2O), 1 ul bracoded adapter, 4ul quick ligase.  The samples were then incubated at room temperature, while rotating for 15 minutes.  After incubation, the samples were washed, as decribed above, in the following buffers: FA lysis buffer; FA lysis buffer+500 mM NaCl; ChIP wash buffer; TE (10 mM Tris pH 8.0, 1 mM EDTA). Spin-X columns were transferred to a new microcentrifuge tube and 100 uL of ChIP elution buffer was added.  Samples were incubated for 10 minutes at 65 degrees C then spun for 1 min at 1500 x g.  Eluted DNA was then transferred to a new microcentrifuge tube and incubated for 10 minutes in a boiling water bath to de-crosslink samples.  The samples were phenol extracted and ethanol precipitated.  The samples were resuspended in 11ul.  To determine cycle numbers for library amplification of each sample, 1ul ChIP DNA was used as a template (diluted 1/8 to fill 3 wells) for Real Time PCR with Illumina primers 1.1 and 2.1 (1 uM ea).  The manual ct was set to 0.1 and 3 was added to the cycle number to determine the amount of cycles for library amplification.   The ChIP DNA was used as a template, using the determined cycle number of : 95 degrees for 30 seconds, 60 degrees for 30 seconds, 72 degrees for 30 seconds.    The following 50ul PCR reaction was used:  5 ul 10x Taq buffer,  8 ul ChIP DNA with ligated adapters, 1.0 ul Illumina primer 1.1 (100 uM), 1.0 ul Illumina primer 2.1 (100 uM), 0.4 ul dNTP mix (25 mM each), 1.5 ul taq, 33.6 ul dH2O.  The libraries were checked on an 8% non-denaturing acrylamide gel.  The smear from 200 bp to 500 bp was cut out and gel purified.  The barcoded libraries were quantified using Qubit and pooled at equimolar concentrations. As Stated above,  Extraction and Library construction occur in the same protocol: Pelleted cells were resuspended in FA lysis buffer containing 2 mg/mL of lysozyme.  Samples were incubated for 30 minutes at 37 degrees C, chilled on ice for at least 5 minutes, and then sonicated for 30 minutes, with 30 s pulses followed by 30 s off.  Samples were spun at maximum speed in a microcentrifuge, the supernatants were removed and diluted to 1 mL lysate per 20 mL original culture volume.  Samples were immunoprecipitated according to the following procedure:  for every 800 uL of chromatin, 25 uL of protein A beads (50% TBS slurry), and 2 uL anti-FLAG antibody (M2) OR 2ul anti-s70 antibody were added.  Samples were incubated on rotisserie rotater for 90 minutes at room temperature.  Samples were then transferred to Spin-X columns (Corning) and washed twice with FA lysis buffer (150 mM NaCl), 3 minutes each wash, rotating at room temperature.  The samples were spun at 4000 rpm after each wash.  The samples were then washed twice in the same way with 10 mM Tris-HCl, pH 7.5. The immunoprecipitated DNA, still attached to protein A beads, were then treated with Quick Blunting Kit (NEB):  The samples were resuspended in the following mixture: 10 ul 10x quick blunting buffer, 10 ul dNTP mix (kit supplied), 80 ul dH2O, 2ul blunt enzyme mix.  The samples were incubated at room temperature, while rotating, for 30 minutes.  The samples were then washed twice with FA lysis buffer (150 mM NaCl), as described above, and then washed twice with 10 mM Tris-HCl, pH 8.0.  The samples were then A tailed, using Klenow (3'->5'exo-), from NEB:  The samples were resuspended in the following mixture: 10 ul 10X NEBuffer 2, 2 ul 100mM dATP (NEB), 88 ul dH2O, 2 ul Klenow.  The samples were incubated at 37 degrees, while rotating, for 30 minutes. The samples were then washed twice with FA lysis buffer (150 mM NaCl), as described above, and then washed twice with 10 mM Tris-HCl, pH 7.5.  Illumina adapters were then ligated to the samples using the NEB Quick Ligation kit.  The samples were resuspended in the following mixture:  100 ul 1x ligase buffer (50 ul 2x buffer + 50 ul dH2O), 1 ul bracoded adapter, 4ul quick ligase.  The samples were then incubated at room temperature, while rotating for 15 minutes.  After incubation, the samples were washed, as decribed above, in the following buffers: FA lysis buffer; FA lysis buffer+500 mM NaCl; ChIP wash buffer; TE (10 mM Tris pH 8.0, 1 mM EDTA). Spin-X columns were transferred to a new microcentrifuge tube and 100 uL of ChIP elution buffer was added.  Samples were incubated for 10 minutes at 65 degrees C then spun for 1 min at 1500 x g.  Eluted DNA was then transferred to a new microcentrifuge tube and incubated for 10 minutes in a boiling water bath to de-crosslink samples.  The samples were phenol extracted and ethanol precipitated.  The samples were resuspended in 11ul.  To determine cycle numbers for library amplification of each sample, 1ul ChIP DNA was used as a template (diluted 1/8 to fill 3 wells) for Real Time PCR with Illumina primers 1.1 and 2.1 (1 uM ea).  The manual ct was set to 0.1 and 3 was added to the cycle number to determine the amount of cycles for library amplification.   The ChIP DNA was used as a template, using the determined cycle number of : 95 degrees for 30 seconds, 60 degrees for 30 seconds, 72 degrees for 30 seconds.    The following 50ul PCR reaction was used:  5 ul 10x Taq buffer,  8 ul ChIP DNA with ligated adapters, 1.0 ul Illumina primer 1.1 (100 uM), 1.0 ul Illumina primer 2.1 (100 uM), 0.4 ul dNTP mix (25 mM each), 1.5 ul taq, 33.6 ul dH2O.  The libraries were checked on an 8% non-denaturing acrylamide gel.  The smear from 200 bp to 500 bp was cut out and gel purified.  The barcoded libraries were quantified using Qubit and pooled at equimolar concentrations.