GSM2544618: MS2_TotRNA; Staphylococcus aureus; RNA-Seq
To purify total RNA, bacterial lysis was performed with FastPrep and the RNA purification followed strictly the procedure described for the FastRNA Pro Blue Kit (MP Biomedicals). For MS2-purified RNA, 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. For both total RNA extracts and MS2-eluted RNAs, 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. The RNAs for total transcriptomics were then treated to deplete abundant rRNAs (Ribozero), and the cDNA libraries were performed using the Random Hexamer approach and sequenced by Fasteris (Switzerland). The standard protocol used is the “TruSeq Stranded mRNA” which is based on the TruSeq Illumina kit and which preserves the information about the orientation of the transcripts and produces reads of 125 nts, which map on the complementary strand. The libraries were sequenced using Illumina Hi-Seq 2500 with High-Output (HO) mode using a V4 chemistry sequencing kit (Illumina).