RIP-seq identification of RNAs enriched on RNA polymerase and the primary sigma factor (sigma A) in Mycobacterium tuberculosis
RIP-seq identification of RNAs enriched on RNA polymerase and the primary sigma factor (sigma A) in Mycobacterium tuberculosis
100 ml 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. 2-3 loops of M. tuberculosis H37Rv cells (grown on Löwenstein–Jensen agar plates for 21 days) were inoculated into Middlebrook 7H9 medium supplemented 10% (v/v) OADC, 0.2% glycerol and 0.05% Tween 80 and cultivated at 35.5°C for 15 days. 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), disrupted by vortexing with 0.1 mm zirconia beads 7x30s with 1 min pauses on ice and centrifuged. 3 mg (protein) of lysates were incubated for 2 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. 14 ul of RNA sample was used for library construction according to the NEXTFLEX® Rapid Directional RNA-Seq Kit. For input samples, 100 ng of RNA was used for library construction. DNA concentrations of IgG control libraries were not measurable, therefore these libraries were not sequenced.