RNA-seq of Pseudomonas aeruginosa PAO1 grown in SCFM2, 6 hour static in 4-well chamber slides, sample 20
Samples were preserved immediately in RNA-later and stored at -80C. Samples were thawed, RNA-later was removed, and samples were resuspended in RNase-free TE buffer containing lysozyme. Samples were incubated for 30 min at 37C for enzymatic lysis, RNA Stat-60 was added, and samples were bead beat 3X for 30 s placing on ice in between each round. Chloroform was added to each sample, samples were mixed and centrifuged to separate phases. The aqueous phase was removed to a new tube and the RNA was precipitated with isopropanol. RNA was fragmented with the NEBNext Magnesium fragmentation module per the manufacturer's instructions. Ribosomal RNA was depleted with the MicrobExpress bacterial mRNA enrichment kit. Sequencing libraries were prepared with the NEBNext Small RNA Library prep kit, and adapter dimers were removed by size selection on a 5% TBE polyacrylamide gel.