Illumina NovaSeq 6000 paired end sequencing
The cell pellets were re-suspended in 500 µL of Disruption solution (50 mM Tris-HCl pH 7.6, 10% sucrose, 5 mM EDTA). The bacteria were transferred to the screw-cap 2 mL tubes with 0.1 mm glass beads (Roth) and disrupted on Fastprep-24 5G (MP Biomedicals) using settings for S. pyogenes (speed 6.0, time 20 s). Phenol-chloroform extraction was performed with 1 mL of Trizol and 0.2 mL of chloroform and the aqueous phase was collected. One more round of extraction was performed with the equal volume of 1:1 acidic phenol-chloroform mixture and the residual amounts of phenol were removed by chloroform extraction. RNA was precipitated by mixing the aqueous phases with the equal volume of 2-propanol, incubation for 1 hour at -20°C and centrifugation for 30 min at 20000 g +4°C. The pellets were washed with 500 µL of 80% ethanol, dried on the bench and dissolved in 100 µL of mQ water. For each sample 10 µg of RNA were treated with Turbo DNase (Thermo Scientific) and RNA was purified with RNA Clean & Concentrator-5 kit (Zymo Research) according to the “Total RNA Clean-up” protocol. Ribosomal RNA was depleted with Pan-Bacteria riboPOOL kit (siTOOLs) and RNA was once again purified with RNA Clean & Concentrator-5. 40 ng of RNA of each sample were used to create the libraries using NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs). The libraries were amplified with 10 cycles of PCR and two times purified with NEBNext Sample Purification Beads to completely remove self-ligated adapters.