ChIP-seq analysis of RNA polymerase and sigma A distribution in Mycobacterium smegmatis
ChIP-seq analysis of RNA polymerase and sigma A distribution in Mycobacterium smegmatis
100 ml of bacterial culture was crosslinked with 1% formaldehyde (final conc.), formaldehyde was added directly to the medium) for 30 minutes at 37 °C in the shaker. Formaldehyde was quenched by glycine (0.125 M final conc.) added for 5 minutes at 37 °C in the shaker. Bacterial cultures were centrifuged for 5 min at 9000 rpm, 4 °C, pellet washed with 10 ml of 1x PBS, bacteria centrifuged again and pellet immediately frozen at -80°C. Mycobacterium smegmatis mc2 155 cells (wt, LK865) were grown at 37 °C in Middlebrook 7H9 medium with 0.2% glycerol and 0.05% Tween 80, and harvested in exponential (OD600 ∼0.5) or early stationary phase (OD600 ∼2.5–3, 24 hrs of cultivation). Bacterial pellet was resuspended in 3 ml of ice cold RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% dexycholate, 0.1% SDS, 50 mM Tris-Cl pH 8.0, 5 mM EDTA) with protease inhibitors cocktail (Sigma-Aldrich, 5 ul/1 ml) and 0.1 M PMSF (5 ul/1 ml) and sonicated 18x10 seconds with 1 min pauses on ice between cycles. Lysate was centrifuged 9000 rpm at 4 °C for 15 min and pellet discarded. 2 mg (protein) of lysates were incubated for 16-18 hours at 4 °C with 20 μl of Protein G plus agarose beads (Santa Cruz) coated with 5 μg mouse monoclonal anti-beta subunit of RNAP antibody [clone 8RB13] (Biolegend), 5 μg of anti-Sigma70 antibody [clone 2G10] (Biolegend), or 5 μg of mouse nonspecific IgG (Sigma-Aldrich) used as a negative control, respectively. The captured complexes were washed twice with RIPA, four times with 100 mM Tris-HCl, pH 8.5, 500 mM LiCL, 1% Triton X-100, 1% deoxycholic acid. Beads with captured complexes were transfered to new eppendorf tube to reduce the background and washed again two times with RIPA and TE buffer. Protein-DNA complexes were eluted with elution buffer (50 mM Tris-Cl pH 8, 0.66 Mm EDTA, 1% SDS) for 10 minutes at 65°C, decrosslinked in the presence of 200 mM NaCl for 5 h at 65°C, treated with 100 µg/ml RNase A for 1 hour at 37°C and 400 µg/ml proteinase K for 30 min at 45°C. DNA was purified with Qiagen PCR purification kit and eluted into 100 ul EB. 40 ul of immunoprecipitated DNA sample or 10 ng of DNA input were used for library construction according to the NEXTflex™ ChIP-Seq Kit manual including the Size-Selection Cleanup step B2.