title: GSM6514649: SP1380 wild-type strain replikate 3; Streptococcus pyogenes; RNA-Seq
library_construction_protocol: RNA was isolated using the RNeasy Plus Kit (Qiagen). Briefly, bacteria were grown to late-logarithmic growth phase in THY. Two volumes of RNAprotect (Qiagen) were added to the cultures and the samples were then centrifuged at 5,000 x g for 25 min at 4°C to pellet cells. RNA was isolated from the dry pellet as per the manufacturer's instructions with an additional mechanical lysis step using Lysing Matrix B tubes (MP Biomedicals) on the FastPrep-2 5G bead beating grinder and lysis system (MP Biomedicals). To ensure complete removal of DNA, the RNA was then further purified using the TURBO DNA-free kit (Thermo Fisher Scientific) according to the manufacturer's instructions. RNAseq analysis was performed at the Australian Centre for Ecogenomics (University of Queensland, Brisbane, Australia). cDNA libraries were prepared from total RNA using TruSeq Stranded Total RNA Library Prep with Ribo-Zero Plus rRNA Depletion kit (Illumina). Sequencing of the cDNA libraries was performed on the NovaSeq 6000 System (Illumina) on a 2x150 bp SP flowcell run generating an average of 20 million reads per sample.