GSM8161199: dR 2; Streptococcus pneumoniae; RNA-Seq
Overnight cultures of S. pneumoniae TIGR4 WT and derivatives were diluted in pre-warmed THY to a final OD600 of 0.05 and incubated at 37°C until OD600 ≈ 0.3. 25 mL of culture was collected, mixed with 1 volume of stop solution (10 mM Tris pH 7.2, 25 mM NaNO3, 5 mM MgCl2, 500 µg/mL Cm) and harvested by centrifugation (10 min, 6000 x g, 4°C). For mRNA decay studies, transcription was stopped by addition of rifampicin (0.5 mg/mL) and nalidixic acid (20 µg/mL) to growing cells (OD600 ≈ 0.3). At the indicated times, 25 mL of culture was collected, mixed with 1 volume of stop solution and harvested by centrifugation in the conditions described above. In both cases, total RNA was extracted using Trizol reagent (Ambion) rRNA was depleted from the samples with the RiboZero kit and sequencing libraries were constructed with the TruSeq kit (Illumina) for bacteria.