RNA-seq of Pseudomonas aeruginosa from human expectorated cystic fibrosis sputum, Sample EM70
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. Ribosomal RNA was depleted and sequencing libraries prepared with the Zymo-seq RiboFree Total RNA Library Prep Kit according to the manufacturer's protocol.