GSM8971708: Aba_sigAb-overexpression_RNAseq_t=5min_rep2; Acinetobacter baumannii; RNA-Seq
Total RNA was extracted using hot phenol organic extraction. Mid-log cells were added to 1.25 mL stop solution (5% water-saturated phenol in ethanol), spun down at 11,000xg for 5 min at 4°C, and pellets were flash frozen in a dry ice:ethanol bath and stored at -80°C. Cells were lysed with lysozyme and SDS, total RNA was purified with phenol, phenol:chloroform, and chloroform extractions followed by ethanol precipitation, and residual DNA was removed with Turbo DNase I treatment. Ribosomal RNA (rRNA) was depleted from the total RNA samples using an RNase H-based protocol. Briefly, DNA oligos complementary to A. baumannii 23S, 16S, and 5S rRNA were annealed to the total RNA samples, RNase H treatment was performed to cleave the annealed rRNA, and the DNA oligos were removed with Turbo DNase I. rRNA-depleted samples were prepared for next-generation sequencing using NEBNext Ultra II Directional RNA Library Prep kit (NEB; E7765S) following manufacturer's protocol.