For genomic DNA (gDNA) extraction, isolates were grown from single colonies in tryptic soy broth (TSB) liquid cultures overnight at 37°C with shaking at 225 rpm. Cells underwent high-molecular-weight (HMW) DNA extraction using enzymatic lysis with lysozyme, lysostaphin, and proteinase K and Qiagen Genomic-Tip columns, as described previously (64 (link)).
For RNA extraction, cultures grown overnight were diluted, grown to late log phase (optical density [OD] of ∼0.80), and stabilized in RNAlater. Total RNA was isolated and purified by using the Qiagen RNeasy minikit. Lysozyme and lysostaphin were used for cell wall degradation, followed by two cycles of 2 min of bead beating with 1 ml of 0.1-mm silica beads in a mini-bead beater (BioSpec), and RNA was eluted in nuclease-free water. Isolated RNA was treated with 1 μl of Baseline Zero DNase (Epicentre) at 37°C for 30 min, and rRNA depletion was performed by using an Epicenter Ribo-Zero magnetic gold kit (Illumina), according to the manufacturer's instructions.
For RNA extraction, cultures grown overnight were diluted, grown to late log phase (optical density [OD] of ∼0.80), and stabilized in RNAlater. Total RNA was isolated and purified by using the Qiagen RNeasy minikit. Lysozyme and lysostaphin were used for cell wall degradation, followed by two cycles of 2 min of bead beating with 1 ml of 0.1-mm silica beads in a mini-bead beater (BioSpec), and RNA was eluted in nuclease-free water. Isolated RNA was treated with 1 μl of Baseline Zero DNase (Epicentre) at 37°C for 30 min, and rRNA depletion was performed by using an Epicenter Ribo-Zero magnetic gold kit (Illumina), according to the manufacturer's instructions.
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