GSM4826369: Serotype 1 A42174 strain MEP; Streptococcus pneumoniae; RNA-Seq
Ribonucleic acid (RNA) from each phase was stabilised by incubating the bacteria in a 5% phenol (Invitrogen) and 95% ethanol solution for 30 minutes on ice. Following stabilisation, the RNA was extracted using a phase separation method using TRIzol® (Invitrogen) (Kroger et al, 2012) RNA libraries for samples 1-3 were assumably prepared for sequencing using standard Illumina protocols. RNA samples 4-6  were fragmented with ultrasound (4 pulses of 30 sec at 4°C) followed by a treatment with antarctic phosphatase and re-phosphorylated with polynucleotide kinase (PNK). Afterwards, the RNA fragments were poly(A)-tailed using poly(A) polymerase and a RNA adapter was ligated to the 5'-phosphate of the RNA. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and M-MLV reverse transcriptase. The resulting cDNA was PCR-amplified to about 20-30 ng/μl using a high fidelity DNA polymerase. The cDNA was purified using the Agencourt AMPure XP kit. The cDNA samples were pooled in equimolar amounts in three different pools. The cDNA in the size range of 150 – 500 bp was eluted from preparative agarose gels. Aliquots of the size fractionated cDNAs were analyzed by capillary electrophoresis.   (Beckman Coulter Genomics) and analyzed by capillary electrophoresis.