GSM8262906: Gentamicin 60 min, input DNA; Escherichia coli; ChIP-Seq
The Cells were harvest by spin at 5,000 g for 3 min, and washed once with 20 ml PBS. The washed cell pellets were then resuspended in 1% paraformaldehyde (Beyotime, P0099) diluted by PBS (5 ml for 40 ml culture of OD600~0.5, with the volume adjusted accordingly), and incubated at 25 °C for 30 min with rotation to perform crosslinking. The fixation reaction was quenched by adding Glycine-HCl (Solarbio, G8200; Sigma, 320331) solution to a final concentration of 125 mM. The fixed cells were collected and washed twice with 10 ml TBS (50 mM Tris HCl, 150 mM NaCl, pH 7.4), and then frozen by liquid nitrogen and stored at -80 °C before use. The thawed pellets were resuspended with 1.1 ml TBS supplemented with proteinase inhibitor (Thermo Fisher, A32955), and sonicated on ice until the resuspensions turned clear. After spin at 12,000 g for 10 min, the supernatant of lysates was transferred to a new tube. Each lysate was split into a DNA input control (0.1 ml) and immunoprecipitation (IP) samples for Gam-3xFLAG ChIP (0.5 ml) and RpoB ChIP (0.5 ml). For the RpoB ChIP experiments, 8 μl of anti-RpoB antibody (Abcam, ab191598) was added to the IP lysate and incubated overnight at 4 °C with rotation. Next, 20 μl of Dynabeads Protein G (Thermo Fisher, 10003D) were added to the IP-RpoB lysate and incubated for 3 h at 4 °C with rotation. The beads were pelleted using a magnetic rack, and washed three times with 0.2 ml of TBS. During the last wash, the resuspension was transferred to a new tube before discarding the supernatant. Elution was performed by resuspending the washed beads in 70 ml of 50 mM Glycine-HCl (pH 2.8). After a 2-min incubation, the eluate was carefully collected. The elution was performed twice, and the eluates were combined. Finally, 15 μl of 1M Tris (pH 8.0) was then added to neutralize the pH. For the Gam-3xFLAG ChIP experiments, 20 μl ANTI-FLAG M2 magnetic beads (Sigma, M8823) were added to another IP lysate and incubated for 2 h at 4 °C with rotation. The beads were then washed and eluted as stated in the RpoB ChIP experiment.  Together with the DNA input control, we incubated the eluates from RpoB ChIP and Gam-3xFLAG ChIP at 65 °C overnight with mild shaking, supplementing with Proteinase K at a final concentration of 0.4 mg/ml. We then extracted the DNA from those samples using the MasterPure Complete DNA/RNA Purification Kit, and measured the concentration with the Qubit 1xdsDNA HS Assay Kit. For library construction, we used the same process as described in the genome re-sequencing, but input 5 ng DNA per sample and scaled down the tagmentation system accordingly.