title: GSM6329867: S.aureus - pD4-19adapted (biol rep 1); Staphylococcus aureus subsp. aureus RN4220; RNA-Seq
library_construction_protocol: 1/10 volume EtOH/Phenol was added to 500 µl of sample and mixed for 1 min. After incubation on ice for 5 min, the samples were centrifuged for 1 min at 20 000 xg at 4°C. The supernatant was discarded, and the pellet was resuspended in 1 ml TRIzol. Each sample was transferred to one screw-cap tube with glass beads and cells were lysed via bead-beating for 2x 6.5 ms/s for 30 s. In between the two runs the cells were kept on ice for 2 min.  200 µl chloroform was added to the samples, mixed and incubated 2-3 min before centrifugation of the samples for 15 min, 12 000 xg, 4°C. The aqueous supernatant was taken, mixed with 500 µl isopropanol and samples were centrifuged for 10 min, 21 000 xg, 4°C. The supernatant was discarded, and the pellet was resuspended in 500 µl 75% EtOH. After centrifugation for 5 min, 20 000 xg, 4°C, the supernatant was discarded, and the pellet was dried at RT. The pellet was resuspended in 100 µl RNA-grade water and RNA was concentrated via the MN RNA clean up kit (Machery-Nagel). RNA samples were DNase I digested (DNase I recombinant, Rnase-free, Millipore Sigma), cleaned up (RNA Clean & Concentrator-5, Zymo Research), quantified (Qubit RNA BR Assay Kit, ThermoFisher) and normalized to 100 ng in 11 µl nuclease-free water. Library preparation was performed according to the Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus Reference Guide. Library concentration was measured with Qubit DNA HS Assay Kit, (ThermoFisher) on a Qubit Fluorometer (invitrogen) and fragment length was assessed with an Agilent 2100 Bioanalyzer (Agilent High sensitivity DNA Kit, Agilent). Samples were equimolarly pooled and sequenced on NextSeq™ 500 High Output Kit v2.5 (75 cycles) flow cell (Illumina) with 1 x 75 bp read length.