GSM8967346: DisrR_BHI_R1; Staphylococcus aureus; RNA-Seq
Bacterial cells were resuspended in lysis Buffer (20 mM Tris-HCl pH 8, 150 mM KCl, 1 mM MgCl2, 1 mM DTT) and broken using the FastPrep apparatus (MP Biomedicals). Crude extract were loaded on a MS2-MBP coated column and purified by affinity chromatography. Eluted RNA were extracted with phenol:chloroform:isoamylalcohol prior to RNAseq analysis. DNase I (0.1 U/ml) treatment was performed 30 min at 37°C. The reactions mixtures were then purified by phenol/chloroform/isoamylalcohol and subsequent ethanol precipitation. RNA pellets were re-suspended in sterile milliQ water. RNA quality and quantity assessments were performed on Agilent Nano Chip on the Bioanalyzer 2100. Ribosomal RNA was depleted from RNA samples using the QIAseq FastSelect rRNA removal kit (QIAGEN), and cDNA libraries were prepared with the NEBNext Ultra II directional RNA library prep kit for Illumina (NEB). Sequencing of cDNA libraries was performed on the NextSeq 2000 system (Illumina).