anti-RNAP_EX_3
Mycolicibacterium smegmatis MC2 155
Protocols: 50 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. 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). 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. 4 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.
isolate
not applicable
replicate
3
ENA-FIRST-PUBLIC
2024-02-09T10:00:07Z
organism
Mycolicibacterium smegmatis MC2 155
organism
Mycolicibacterium smegmatis MC2 155
ENA-LAST-UPDATE
2024-02-09T10:00:07Z
growth condition
exponential phase
scientific_name
Mycolicibacterium smegmatis MC2 155
ENA-FIRST-PUBLIC
2024-02-09
ENA-LAST-UPDATE
2024-02-09