RIP-seq identification of RNAs enriched on RNA polymerase and the primary sigma factor (sigma A) in Bacillus subtilis
RIP-seq identification of RNAs enriched on RNA polymerase and the primary sigma factor (sigma A) in Bacillus subtilis
150 ml exponential phase and 50 ml stationary phase cells were pelleted and washed in lysis buffer (20 mM Tris–HCl pH 7.9, 150 mM KCl, 1 mM MgCl2, 1 mM dithiothreitol (DTT) and frozen in -70°C. Bacillus subtilis 168 trp+ BaSysBio cells (LK2711) were inoculated into 200 ml of LB medium at OD600 0.03 and grown at 37°C. Bacteria were harvested in exponential (OD600 ∼0.25) or stationary phase (OD600 ∼3–4, 7-8 hrs of cultivation). Cells resuspended in lysis buffer (20 mM Tris–HCl pH 7.9, 150 mM KCl, 1 mM MgCl2, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), supplemented with Calbiochem Protease Inhibitor Cocktail Set III protease inhibitors), sonicated 15 × 10 s with 1 minute pauses on ice and centrifuged. 1.5 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), 2.5 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 four times using 20 mM Tris–HCl pH 7.9, 150 mM KCl, 1 mM MgCl2, finally resuspended in 200 μl 1% SDS, 150 mM KCl, 20 mM Tris–HCl pH 7.9, 1 mM MgCl2 and incubated on a rotating platform with 200 μl acidic phenol (pH∼3):chloroform (1:1) for 15 minutes. Eluted RNA was precipitated with ethanol, dissolved in water and DNase treated (TURBO DNA-free Kit, Ambion). Inputs are total RNA samples isolated from the bacterial lysates. 10% of lysate that was used for one immunoprecipitation was diluted in 1% SDS, 20 mM Tris–HCl pH 7.9, 150 mM KCl, 1 mM MgCl2 to final volume 200 μl and RNA was isolated with the same protocol as the immunoprecipitated RNA. 20 ul of RNA sample was used for library construction according to the NEXTFLEX® Rapid Directional RNA-Seq Kit. DNA concentrations of IgG control libraries were not measurable, therefore these libraries were not sequenced.