RNA-seq of Pseudomonas aeruginosa from human expectorated cystic fibrosis sputum sample EM6
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 and lysostaphin. Samples were incubated for 30 min at 37C for enzymatic lysis, RNA-bee was added, and samples were bead beat 3X for 30 s placing on ice in between each round. Isopropanol 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 ethanol. Ribosomal RNA was depleted with an Illumina RiboZero Gold (bacteria) kit and RNA was fragmented with the NEBNext Magnesium fragmentation module per the manufacturer's instructions. 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.