Rneasy protect bacteria mini kit

Planktonic cultures were generated by overnight incubation with shaking at 180 rpm of a single colony transferred to 10 ml of BHI media. The early stationary growth phase was prepared by the dilution of overnight cultures to an OD₆₀₀ of 0.1 (corresponding to approximately 10⁶ cells/mL) and the transfer of 1 mL to Erlenmeyer flasks containing 10 ml of BHI media in triplicate. The cultures were grown with shaking at 180 rpm to an OD_600nm of 0.8, which corresponds to the early stationary growth phase (Figure S1), and then stabilized by treatment with RNA Protect (RNeasy Protect Bacteria Mini Kit, Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. The preparation of biofilms in six-well plates and the extraction of RNA were performed as described [19 (link)].
RNA was purified using the RNeasy Protect Bacteria Mini Kit (Qiagen) according to the manufacturer’s protocol for enzymatic lysis and the proteinase K digestion of bacteria, with the lysis time extended to 30 minutes. The samples were treated twice with RNAprotect for 15 minutes. Turbo DNase (Ambion) treatment was used to degrade DNA. The integrity of the RNA samples was evaluated by Qubit ™ Fluorometric Quantitation (Thermo-Fisher Scientific, Waltham, MA, USA) and treated with Ribo-Zero™ rRNA Removal Kit (Bacteria) (Illumina, San Diego, CA, USA) to remove rRNA prior to sequencing

Purified RNA was extracted from diluted overnight broth cultures using the ClaremontBio RNAexpress with OmniLyse® HL Kit and DNAexpress columns (Claremont BioSolutions, LLC, Upland, CA) or the Qiagen RNeasy® Protect Bacteria Mini Kit (QIAGEN, Inc., Valencia, CA), according to the manufacturer’s guidelines. The quantity of nucleic acid was assessed by fluorometric measurement using the Qubit™ 4 Fluorometer (Invitrogen, Carlsbad, CA), and the quality was evaluated using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). Crude lysates and plate enumerations were prepared by aliquoting diluted overnight broth culture at mid-log (OD₆₀₀ absorbance ~ 0.2–0.3) and serially diluting tenfold in nuclease-free water and 1 × phosphate-buffered saline (PBS), respectively. To prepare crude lysates, each serially diluted culture in nuclease-free water was lysed by mechanical disruption using an OmniLyser (Claremont BioSolutions, LLC). For plate enumerations, a 10 µl droplet of each serially diluted culture in 1 × PBS was plated on fresh solid LB agar overnight at 37 °C for Citrobacter species, Escherichia species, Listeria species, Salmonella species, and Shigella species strains and 28 °C for Bacillus species, Enterobacter species, and Pseudomonas species.