Bacterial cultures were rapidly cooled on ice, centrifuged, and subjected to disruption with 0.1 mm zirconia beads in a Bead Beater homogenizer (BioSpec Products, Bartlesville, OK, USA) as previously described (Rustad et al., 2009). Total RNA was isolated through phenol-chloroform extraction, treated with Turbo DNase (Life Technologies, Gaithersburg, MD, USA) to remove traces of genomic DNA, purified with the RNeasy Mini Kit (Qiagen, Venlo, The Netherlands). RNA samples were depleted of 16S and 23S rRNA using NEBNext® rRNA Depletion Kit (Bacteria) (NEB). Sequencing libraries were generated using the resulting ribosomal transcript-depleted RNA and NEBNext Ultra II Directional RNA Library Prep Kit (NEB) according to the manufacturers' protocol.