GSM8578254: ST615 LSP; 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, RNA was extracted using a phase separation method using TRIzol® (Invitrogen). Briefly, the cells were centrifuged and re-suspended in TRIzol® to allow bacterial lysis. The lysed bacteria were mixed with chloroform to separate the RNA from DNA, lipids and proteins. Once the aqueous phase was collected (RNA), the sample was washed and the RNA was precipitated using ethanol. Finally, the RNA was re-suspended in DNase/RNase free distilled water (Invitrogen). The RNA extraction was done in an RNase-free area and using RNase-free materials to avoid RNA degradation. The quantity of RNA was determined using the Qubit® Fluorometer (Thermo Scientific). A 2100 Bioanalyzer (Agilent Technologies, Stanta Clara, USA) was used to assess the quality of the RNA. RNA libraries for samples 1-5 were assumably prepared for sequencing using standard Illumina protocols. RNA samples 6-20  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.