GSM1533478: lpxA control 60min R2; Acinetobacter baumannii ATCC 19606 = CIP 70.34 = JCM 6841; RNA-Seq
Approximately 75 mL of each culture was transferred to sterile Falcon tubes and cells harvested by centrifugation (4,000 × g for 10 min) at 4°C. The supernatant was removed, and cells were resuspended in 1 mL of RNAlater RNA Stabilization Reagent (Qiagen) and stored at 4°C for up to 4 days. Cell samples, previously resuspended in RNAlater,were centrifuged at 5,000 × g for 10 min, and the supernatant discarded. The pellet was then resuspended in 200 µL of 10 mg/mL lysozyme (Sigma) in TE buffer to which 1 µL Protector RNase Inhibitor (Roche) had been added. The solution was vortex-mixed for 10 sec and incubated at room temperature for 10 min, with further vortex mixing for 10 sec every 2 min. RNA was purified and concentrated using the RNeasy MinElute Cleanup Kit (Qiagen), according to the manufacturer's RNA cleanup protocol, including an on-column DNase treatment using the RNase-free DNase Set (Qiagen). An additional step was performed after the addition of 700 µL of Buffer RLT to the extracted RNA, as follows; samples were vortex-mixed vigorously and centrifuged at 5,000 × g for 5 min to pellet any precipitate. The supernatant was transferred to a fresh tube and RNA precipitated by the addition of three volumes of ethanol. For further elimination of contaminating DNA, the isolated RNA was treated again with the RNase-free DNase Set (Qiagen), according to the manufacturer's protocol. The construction of the cDNA library from purified RNA samples was performed using the Truseq RNA Preparation Kit (Illumina). Ribosomal RNA (rRNA) was removed from the samples using the Ribozero kit (Epicentre). Approximately 300 ng of rRNA-depleted RNA was used as input, following the manufacturer's protocol (low throughput).
GEO Accession
GSM1533478