AB 5500xl Genetic Analyzer paired end sequencing; The transcriptome of TRPV1-positive sensory neurons revealed by subgroup-elimination transcriptomics
The transcriptome of TRPV1-positive sensory neurons revealed by subgroup-elimination transcriptomics
Lumbar dorsal root ganglia were isolated from 8 weeks old Spargue Dawley rats Doral root ganglia were isolated from three rats were split into half and treated with solvent DMSO (0.1%) or RTX (100 nM) for 30 min. Three replicate experiments were performed at the same time on different days resulting in 6 samples (2 samples each from 3-rat pools '120726', '120727' and '120730').  For the RTX treatment, dorsal root ganglia were incubated in 5 ml MEM containing collagenase P (Roche, Penzberg, DE) (0.1 U/ml, 1 h, 37 degrees C, 5% CO2), dissociated by trituration with fire-polished Pasteur pipettes, and then treated with the solvent DMSO (0.1%) or RTX (100 nM). The cells were spun down (100 g, 5 min), resuspended in 1 ml Hank's buffered salt solution (pH 8.0) containing 0.025% EDTA and 245 U/ml trypsin (Worthington, Lakewood, NJ), and incubated for 4 min at 37 degrees C in a water bath. Trypsination was stopped by the addition MgSO4 (400 uM final conc.). The cell suspension was then loaded onto BSA gradients (15% BSA in Hank's buffered salt solution) and centrifuged (120 g, 8 min). The pellet was frozen in liquid nitrogen and stored at -80 degrees C for RNA isolation. RNA isolation was performed with NucleoSpin RNA/Protein kits (Macherey-Nagel, Dueren, Germany) including on column DNase treatment. Ribosomal RNA was removed from 5 ug total RNA per sample using Ribo-Zero rRNA Removal Kit (Epicentre Biotechnologies, Madison, WI, USA).
Experimental Factor: cell type
dorsal root ganglia cell depleted of TRPV1 expressing neurons