title: RNA-seq of Pseudomonas aeruginosa from human induced cystic fibrosis sputum, Sample EM65
design: 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. 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. Ribosomal RNA was depleted with the NEBNext rRNA Depletion Kit with Human/Mouse/Rat and Bacterial probes mixed 1:1. 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.