hilD ChIP Seq
hilD ChIP Seq
2 x 2mL cultures were set up in LB + 0.3 M NaCl and grown overnight at 37 degrees C/250rpm.  Overnight cultures were diluted 1:100 into 16 x 20mL subcultures and grown to an OD600 of between 0.9 and 1.0.   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. 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, 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. 20 uL of each chromatin sample was set aside to be used as an input control.  Samples were immunoprecipitated according to the following procedure:  for every 800 uL of chromatin, 25 uL of protein A beads and 2 uL anti-FLAG antibody (M2) were added and samples were incubated on rotisserie rotater for 90 minutes at room temperature.  Samples were spun for 1 min in a microcentrifuge at 1500 x g and supernatant discarded.  Protein A beads were resuspended in FA lysis buffer and transferred to a Spin-X spin column.  Columns were incubated for 3 min at room temperature on rotisserie rotater, spun for 1 min at 1500 x g and supernatant was discarded.  Columns were washed with the following buffers, each with a 3 minute incubation at room temperature on rotisserie rotater, spin for 1 min at 1500 x g and discard supernatant:  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 and input DNA (diluted to 100 uL with ChIP Elution buffer) were then transferred to a new microcentrifuge tube and incubated for 10 minutes in a boiling water bath to de-crosslink samples.  ChIP samples and input DNA were then cleaned up using the Qiagen PCR Purification Kit, eluting in 50 ul. 32 equivalent samples were pooled, ethanol precipitated, and resuspended in 11 uL water.  DNA was then treated using the NEB Quick Blunting Kit using 1 uL of Blunting Enzyme Mix (NEB) according to the manufacturer's instructions.  Samples were incubated for 30 min at room temperature, followed by inactivation of the enzyme by heating samples to 70  degrees C for 10 minutes.  Once cooled, samples were treated with RNase A, incubated for 15 min at 37  degrees C and purified using the Qiagen PCR Purification Kit, eluting in 11 uL.  Samples were then poly-A tailed using Terminal Transferase (10.8 uL DNA, 2 uL 2.5 mM CoCl2, 2 uL 10X terminal transferase buffer, heat at 95  degrees C for 5 minutes, cool at 4  degrees C, add 4 uL 50 uM dATP, 0.2 uL BSA, 0.75 uL water and 0.25 uL Terminal Transferase and incubated at 37  degrees C for 1 hour, 70  degrees C for 10 min, cooled to 4  degrees C), and blocked using biotin-ddATP (heat 20 uL A-tailing reaction at 95  degrees C for 5 min, cool to 4  degrees C, add 1 uL 2.5 mM CoCl2, 1 uL 10X Terminal Transferase buffer, 0.5 uL 200 uM biotin-ddATP, 7.25 uL water, 0.25 uL Terminal Transferase, incubated at 37  degrees C for 1 hour, 70  degrees C for 20 min, cool to 4  degrees C)
Experimental Factor: strain
14028s HilD-FLAG3
Experimental Factor: immunoprecipitate
M2 anti-FLAG monoclonal Ab