GSM3454202: MS2-RsaI_R3; 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. 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 1h 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. RNAs were treated to deplete abundant rRNAs, and the cDNA libraries were performed using the ScriptSeq complete kit (bacteria) from Illumina. The libraries were sequenced using Illumina MiSeq with a V3 Reagent kit (Illumina), which preserves the information about the orientation of the transcripts and produces reads of 150 nts, which map on the complementary strand. Each RNA-seq was performed at least in duplicates.
GEO
GSM3454202