RNA/DNA hybrid dot blot

Single colonies were grown overnight in LB at 30°C. Cultures were diluted to an OD₆₀₀ of 0.05 into fresh LB medium supplemented with 1 mM IPTG for both B. subtilis morning and overnight inductions. For the FL-PcrA overexpression experiment, overnight cultures were induced the next morning at the time that they were diluted. For the CTD experiments, induction was started while inoculating the single colonies for the overnight growth. Cultures were grown until an OD₆₀₀1.1–1.2 and genomic DNA was purified using the DNeasy Blood and Tissue (Qiagen) or the GenElute Bacterial Genomic DNA Kit (Sigma-Aldrich) following manufacturer’s instructions. gDNA was quantified by Nanodrop and a fraction of DNA was treated with 5 U RNase H (NEB) for 1 hr at 37°C. gDNA serial dilutions and the RNase treated gDNA were spotted on a positively charged Hybond-N+ nylon membrane (Amersham) using a dot-blot apparatus. The DNA was probed with the S9.6 antibody (1:1000 dilution, Millipore) in 1% BSA/TBST overnight at 4°C after UV-crosslinking (0.12 J/cm²) and blocking the membrane with 5% milk/TBST for 1 hr at RT. An anti-mouse HRP antibody (1:10000 dilution, Santa Cruz Biotechnology) was used as secondary antibody. Images were acquired with Odyssey Fc (Li-COR Biosciences). DNA loading was calculated by staining with 0.05% methylene blue in 0.5 M sodium acetate buffer (pH 5.2) after washing the membrane with 5% acetic acid as described previously (Ko et al., 2010; Raghunathan et al., 2018). Images were quantified using ImageQuant (Cytiva). Error bars show the SEM of at least three independent experiments.