GSM5965599: USA300_human_serum_medium_Nanopore; Staphylococcus aureus; RNA-Seq
RNA extraction was performed using the guanidium thiocyanate (GTC) method. Briefly, 5 ml of cell culture was harvested at exponential phase by centrifugation. Cell pellets were resuspended in 100 µl GTC-Phenol (pH 7; 1:1 ratio) and lysed by vortexing the cells for 5 min with 100 µl of Zirconia beads (0.1 mm; Biospec products 11079101z). Subsequently, 550 µl of GTC was added and the mixture was vortexed for several minutes and incubated at 65ºC for 10 minutes and then 10 minutes on ice. Phases were separated by adding 300 µl chloroform isoamylalcohol (24:1) and 1/10th of a volume of 3M NaAc (pH 5.2) followed by rigorous vortexing. After 5 minutes of centrifugation in an Eppendorf centrifuge (14k rpm), the RNA was purified from the upper phase via two additional rounds of phenol-chloroform extraction and precipitated with 3 volumes of 96% cold ethanol. Nanopore cDNA libraries were prepared using total RNA isolated from wild type USA300 grown in TSB or human serum. Total RNA was DNase I (TURBO DNase; Thermo Fisher) treated for 1 hour at 37°C in the presence of 2 U of SUPERasin. RNA was subsequently poly(A) tailed using E. coli poly(A) polymerase (NEB) following the manufacturer's instructions. Afterwards, rRNA was removed using the NEBNext rRNA Depletion Kit (Bacteria) according to the manufacturer's instructions. After each enzymatic step pior to the use of Nanopore cDNA kit, RNA was purified using RNAClean XP beads (Beckmann Coulter). cDNA barcoded libraries were prepared with Nanopore cDNA-PCR Sequencing kit (SQK-PCS109).  The cDNA was purified without size selection in order not to exclude sRNAs. The pooled barcoded libraries were sequenced on MinION using an R9 flow cell.