GSM2532794: FstpAD1 high level exposure; Enterococcus faecalis; RNA-Seq
Cell pellets were resuspended in Bacteria RNA Protect (Qiagen) and incubated at room temperature for 5 minutes. Cells were then pelleted and stored at -80⁰C. Once all samples were collected, pellets were thawed and resuspended in 200µl Tris/EDTA/ mutanolysin/lysozyme solution (10 mM Tris [pH 8.0], 1.0 mM EDTA [pH 8.0], mutanolysin (500U ml-1), lysozyme (30 mg ml-1)) and incubated at 37⁰C for 10 minutes. To the resuspensions, 700 µl of Buffer RLT (Qiagen RNeasy Mini Kit) with 10 µl β-ME was added and vortexed to mix. RNA was then purified using the Qiagen RNeasy Mini Kit according to the manufacturer’s instructions. Total RNA from biological replicates was treated with TURBO DNase (Ambion / ThermoScientific, Waltham, MA) using the rigorous protocol according to the manufacturer’s instructions. DNase was removed from treated samples using the RNA Clean & Concentrator-25 (Zymo Research). mRNA was enriched by rRNA removal using the MICROBExpress Bacterial mRNA Purification Kit (Ambion/ThermoScientific) according to the manufacturer’s instructions. University of Minnesota Genomic Center (UMGC) performed QC and library preparation.  mRNA quantified using fluorimetry (RiboGreen assay) and RNA integrity assessed using capillary electrophoresis (e.g., Agilent BioAnalyzer 2100), generating an RNA Integrity Number (RIN). For a sample to pass QC, mass must be ≥ 1 ug, with a RIN > 8.  Library created using TruSeq RNA V2 kit.  Briefly, cDNA was generated using reverse transcriptase, fragmented and blunt-ended, and ligated to indexed (barcoded) adapters. (Barcode Data: RF_5_pCIE_A, AGTCAA; RF_5_pCIE_B, AGTTCC; RFE_1_A, ACAGTG; RFE_1_B, GCCAAT; RFE_5_A, CAGATC; RFE_5_B, ACTTGA; RFA_5_A, GATCAG; RFA_5_B_TAGCTT). The library was gel size selected to generate inserts of ~200 bp.
GEO Accession
GSM2532794