Illumina HiSeq 2500 sequencing; RNA-Seq of rat dorsal root ganglions treated with paclitaxel against untreated controls
RNA-Seq of rat dorsal root ganglions treated with paclitaxel against untreated controls
Rat lumbar DRG (L4-L5) were collected at day14 after the initiation of paclitaxel or vehicle treatments (n=4 rats). Specifically, DRG tissue were rapidly removed under isoflurane anesthesia and snap frozen. First, we separated the spine at L4-L5 segment. The dorsal skin and musculature were cut either side of the spine. The most inferior ribs were used as a landmark. T13 was located just inferior to the ribs, and its next section was lumbar 1. The iliac crest was also used as an anatomical marker for the location of the 4th and 5th lumbar spine. Next, we opened the spine, separated the spinal cord, then opened the vertebral foramina of both sides, and carefully pulled out the DRGs. Total RNA was extracted from rat L4-5 DRGs using Trizol reagent kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturerâ€™s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel electrophoresis. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo(dT) beads, while prokaryotic mRNA was enriched by removing rRNA by Ribo-Zero TM Magnetic Kit (Epicentre, Madison, WI, USA).  The enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcription into cDNA with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kit (Qiagen, Venlo, The Netherlands), end repaired, poly(A) added, and ligated to Illumina sequencing adapters.