Zymo rna clean concentrator columns

Two volumes of RNA-protect Bacterial Reagent (Qiagen Sciences, Inc., Germantown, MD) were added to one volume (1.5 ml) of culture (approximately 10⁹ CFU/ml) grown statically in M9GT ± 1% glycine at 37°C for 18 h. Cells were incubated at room temperature for 5 min and harvested by centrifugation (5,000 × g, 10 min, 10°C). Total RNA was extracted using the RNeasy Mini Kit (Qiagen Sciences) with an additional on-column DNaseI digestion following the manufacturer's protocol. For transcriptomic analysis, a higher yield of total RNA was required, and thus the RNeasy Midi Kit (Qiagen Sciences) was used. DNA and ribosomal RNA (rRNA) were removed from the extracted RNA using the TURBO DNA-free kit (Thermo Fisher Scientific) and Ribo-Zero rRNA Removal Gram-Negative kit (Illumina, Inc., San Diego, CA). The rRNA-depleted RNA samples were concentrated and purified using Zymo RNA Clean & Concentrator columns (Zymo Research, Irvine, CA). Concentration and quality of RNA were determined using the RNA Nano-chip and the RNA Pico-chip on an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA) for transcriptomic analysis, or a Nanodrop ND-1000 UV-visible wavelength spectrophotometer (NanoDrop Technologies, Wilmington, DE) for RT-qPCR experiments.