GSM4224646: WT M9/rdm/glu stationary phase, ChIP replicate 1; Escherichia coli; ChIP-Seq
Frozen cell pellets were resuspended in 1x IPOD lysis buffer (10 mM Tris HCl, pH 8.0; 50 mM NaCl) containing 1x protease inhibitors (Roche Complete Mini, EDTA free) and 52.5 kU/mL of ready-lyse (Epicentre); 600 uL per pellet (stationary phase cells were diluted 10x prior to lysis due to the much higher biomass of those pellets). We incubated the resuspended pellet for 15 minutes at 30 C, and then placed it on ice. We then sonicated the cells using a Branson digital sonicator at 25% power, using three 10 second bursts with 10 second pauses between bursts. The cells were maintained in a wet ice bath throughout sonication. We then performed a calibrated DNA digestion to sub-200 bp fragments, by adding to the sonicated lysates 60 ug RNase A (Thermo Fisher), 6 uL DNase I (Fisher product #89835), 5.4 uL 100 mM MnCl2, and 4.5 uL 100 mM CaCl2, and then incubating on ice. While the appropriate digestion time must be calibrated for each particular sample type and batch of DNase, 30 minutes of digestion proved appropriate for all samples here. Reactions were quenched after completion by the addition of 50 uL 500 mM EDTA (pH 8.0), typically yielding 50-200 bp fragments. Samples were clarified by centrifugation for 10 minutes at 16,9000xg at 4 C. After clarification, the digested lysates were mixed 1:1 with 2x IP buffer (200 mM Tris, pH 8.0; 600 mM NaCl; 4% Triton X-100; 2x Roche Complete EDTA-free protease inhibitors), and then kept on ice for no more than a few hours prior to antibody addition. We added 10 microliters of purified anti-E. Coli RNA polymerase antibody (Neoclone WP023), and incubated overnight with rocking at 4 C. Near the end of the incubation period, we resuspended an aliquot of 50 microliters of protein G dynabeads (Invitrogen) and equilibrated the protein G beads with 1x IP buffer lacking protease inhibitors. The bead aliquot was added to the antibody-lysate mixture, and then incubated 2 hours with rocking at 4o C. The bead-antibody-target complexes were subsequently subjected to the following series of washes, with 1 mL used per wash. All washes were at room temperature, and involved manual resuspension of the beads in the new wash buffer followed by immediate re-separation: 1x Wash buffer A (100 mM Tris, pH 8.0; 250 mM LiCl; 2% Triton X-100; 1 mM EDTA); 1x Wash buffer B (100 mM Tris, pH 8.0; 500 mM NaCl; 1% Triton X-100; 0.1% sodium deoxycholate; 1 mM EDTA);  1x Wash buffer C (10 mM Tris, pH 8.0; 500 mM NaCl; 1% Triton X-100; 1 mM EDTA); 1x TE (10 mM Tris, pH 8.0; 1 mM EDTA). The antigens were subsequently eluted by adding 500 microliters of elution buffer (composition described above) and incubating 30 minutes at 65 C, with vigorous vortexing every 5-10 minutes, the protein G beads removed, and the remaining supernatant reverse crosslinked overnight at 65 C. After allowing the samples to cool to room temperature, we then added 100ug of RNase A (Thermo-Fisher), incubated 2 hours at 37 C, then added 200ug of proteinase K (Fermentas) and incubated an additional 2 hours at 50 C. DNA was then recovered via standard phenol-chloroform extraction and ethanol precipitation, following protocols from (Ausubel, 1998). We used Glycoblue (Ambion) as a co-precipitant, NaCl as a precipitating salt (due to the presence of SDS in our solution), and washed with ice-cold 95% ethanol to avoid loss of low molecular weight DNA. NEB Nebnext DNA kit, but with a spin column replacing the first bead cleanup