nucleic acid extraction protocol
Pelleted cells were resuspended in FA lysis buffer containing 2 mg/mL of lysozyme. Samples were incubated for 30 minutes at 37 °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.
normalization data transformation protocol
Cy3 or Cy5 intensitites were determined using Agilent Technologies image-analysis software with the local background correction option selected
array scanning protocol
For data collection, we used an Agilent Technologies microarray scanner
nucleic acid hybridization to array protocol
DNA was hybridized to microarrays in an Agilent Technologies microarray chamber by using microarray buffer (1MNaCl, 50 mM MES pH 7, 20% formamide, 1% Triton X-100) and rotated at 55° over 60 h. The arrays were then washed with Wash 1 [6, standard saline phosphate, EDTA (0.18 M NaCl, 10 mM phosphate, pH 7.4, 1 mM EDTA] (SSPE), 0.005% N-lauryl sarcosine] and Wash 2 (0.06% SSPE, 0.18% polyethylene glycol 200), both for 5 min at room temperature
nucleic acid labeling protocol
ChIP DNA (8 samples combined) or input DNA was amplified with Klenow, random hexamer, and Cy3-dCTP or Cy5-dCTP, and purified using a Qiaquick column
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 IgG sepharose beads 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. 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°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 MinElute PCR Purification Kit, eluting in 10 ul.
Cells were transferred to a prewarmed flask at 50 °C for 10 minutes before harvesting
2 mL cultures were set up in LB and grown overnight at 30 °C/250rpm. Overnight cultures were diluted 1:100 into 40 mL and grown to an OD600 of between 0.3 and 0.6 before heat shock. 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 °C.