GSM1412100: t0; Enterococcus faecalis; RNA-Seq
Frozen pellets were thawed, resuspended in 0.4 ml of RNase-free TE containing 50 mg/ml lysozyme and 1000 U/ml mutanolysin, homogenized using a hand-held motorized homogenizer with RNase-free pellet pestles (Fisher Scientific, Pittsburgh, PA), and incubated for 10 min at 37°C.  Each sample was then split in half and extracted in duplicate with the RNeasy Mini Kit (Qiagen Inc.) according to the manufacturer’s instructions with an added homogenization step using the QIAshredder (Qiagen Inc.) immediately after the addition of Buffer RLT.  RNA was eluted from each column with two 30 μl volumes of RNase-free water, and total RNA from duplicate extractions of each sample were pooled together.  All OG1RF(pCF10) RNA eluates obtained from a single rabbit were further pooled.Contaminating DNA was removed using a TURBO DNA-free kit (Ambion, Austin, TX) following the rigorous protocol as directed by the manufacturer. DNase-treated RNA was ethanol precipitated in the presence of a glycogen carrier and resuspended in RNase-free TE.  RNA harvested from rabbits was first treated with MICROBEnrich (Ambion) followed by MICROBExpress (Ambion), according to the manufacturer’s instructions, to deplete eukaryotic RNA and bacterial rRNA, respectively.  RNA from the infection inoculum was treated with MICROBExpress only. Library creation and sequencing were performed by the University of Minnesota Genomics Center.  Indexed libraries were created using the TruSeq RNA Sample Preparation Kit v2 (Illumina, Inc., San Diego, CA) according to the manufacturer’s instructions.  Indexed libraries were size selected to 320 bp +/- 5% using a LabChip XT instrument (Perkin Elmer, Waltham, MA).  Prepared libraries were hybridized to paired end flow cells, clonally amplified on an Illumina cBot, and sequenced using Illumina SBS chemistry in a 100 bp paired end run on an Illumina HiSeq 2000 instrument.
GEO Accession
GSM1412100