title: GSM8397014: LND_0_rep3; Staphylococcus aureus; RNA-Seq
library_construction_protocol: The phenol-chloroform method was used for total RNA extraction. Cell pellets were thawed on ice and suspended in 750μl Tris EDTA (TE) buffer (Sigma Aldrich, Missouri – USA). Cell suspensions were lysed in Lysing Matrix E tubes on a FastPrep machine (both from MP Biomedicals, Irvine, California – USA) at a maximum force of 6.5 for two cycles of 30 sec each. After a 30 sec centrifugation at 4° C, 500μl of the supernatant was transferred into a fresh 2 ml tube to which 60μl of 10% w/v sodium dodecyl sulphate (SDS) was added with mixing. The tubes were incubated at 640C for 2 min followed by addition of 66μl of 1M Sodium acetate (NaOAc) with mixing. Roti‐Aqua phenol (750μl per sample) (Carl Roth, Baden-Wurttemberg, Germany) was added to the samples followed by mixing. The tubes were incubated for 10 min at 64°C with inversion at 2-min intervals followed by centrifugation for 15 min at 4°C. The aqueous portion was added to PLG heavy tubes (MP Biomedicals, Irvine, California – USA) containing 750μl chloroform followed by shaking for 2 min. The tubes were centrifuged for 12 min at 15°C. RNA was precipitated with 600μl isopropanol, washed with 500μl ice cold 70% ethanol (EtOH), air-dried at room temperature, and eluted on ice with 30μl of RNAse-free water (Ambion, Austin, Texas, USA). A Nanodrop spectrophotometer (DeNovix Inc, Wilmington, Delaware – USA) was used for quantification and quality assessment and RNA extracts were frozen at ‐80°C. All centrifugation steps were performed at 13,000 rpm on a 5424R centrifuge (Eppendorf, Hamburg, Germany). RNA extracts were subjected on DNAse digestion to eliminate potential DNA contamination. Ribosomal RNA (rRNA) depletion was performed with RiboCOP‐Meta rRNA depletion (Vienna, Austria) following manufacturer guidelines. Briefly, rRNA-specific magnetic beads are mixed with total RNA, and the mixture denatured where beads are allowed to bind to rRNA followed by magnetic separation and purification of rRNA-free RNA. Lexogen's Corall Library Prep (Vienna, Austria) kit and protocol was used following manufacturer recommendations. In this fragmentation-free, Illumina-compatible library preparation protocol, proprietary Displacement Strand Priming (DSP) is used for reverse transcription followed by 3' Linker Oligo ligation, second strand complementary DNA (cDNA) generation and PCR amplification of the cDNA libraries. All cleaning steps in the protocol are performed using magnetic bead-based approaches. Libraries were checked for quality with Bioanalyzer RNA assay. The pooled sequencing library (1.8 pM) was loaded onto an Illumina NextSeq 500 MidOutput machine (130 M with 1 x 150 cycle single-end sequencing).