GSM3591576: PS2_minus_TEX; Staphylococcus epidermidis; RNA-Seq
Cells were disrupted mechanically using Lysing Matrix E glass bead tubes and the FastPrep® Instrument (MP Biomedicals) and total RNA was extracted using the hot phenol technique. In brief, the supernatant of the disrupted cells was mixed with SDS (1 %), sodium acetate (pH 5.5, 0.1 M) and phenol (1:1) and incubated at 64°C in a water bath for 10 min. The aqueous phase was then transferred to PLG tubes (phase-lock gel tubes, cat. no. 2302830, 5 Prime) and after chloroform extraction (1:1), the aqueous phase was again transferred to fresh tubes and the RNA precipitated with 4X vol. ethanol/3 M sodium acetate pH 6.5 (30:1 mix) at  20°C overnight. Pelleted RNA was washed with 70 % ethanol and finally solved in RNase-free water and remaining genomic DNA was eliminated by DNase I treatment. For depletion of processed transcripts, equal amounts of RNA were incubated with Terminator 5'-phosphate-dependent exonuclease (TEX) (Epicentre #TER51020). The transcripts were not fragmented in order to get mainly sequencing reads of the 5'-end of the transcripts. Libraries for Illumina sequencing of cDNA were constructed by vertis Biotechnology AG, Germany (http://www.vertis-biotech.com/) as described previously (Berezikov et al.,2006), but omitting the RNA size-fractionation step prior to cDNA synthesis. Equal amounts of RNA samples were poly(A)-tailed using poly(A) polymerase. Then, the 5'-triphosphates were removed by applying tobacco acid pyrophosphatase (TAP) resulting in 5'-monophosphate. Afterwards, a RNA adapter was ligated to the 5'-phosphate of the RNA. First-strand cDNA was synthesized by an oligo(dT)-adapter primer and the M-MLV reverse transcriptase. In a PCR-based amplification step using a high fidelity DNA polymerase the cDNA concentration was increased to 20-30 ng/µl. The resulting cDNA libraries were sequenced using a HiSeq 2500 machine (Illumina) in single-read mode and running 100 cycles.