GSM3354698: Vehicle_3d_NU_OG_Rep5; Rattus norvegicus; RNA-Seq
Livers were harvested 24 hours after the last dose as 100 mg punches using six millimeter disposable biopsy punches. An appropriately staggered schedule was employed so that the harvest times were accurate to ±30 min of the designed dose-to-harvest interval.  Automated RNA isolation was performed according to the manufacture's protocol using the RNeasy Kit from Qiagen (Germantown, MD). The sequencing library for the rat liver RNA samples was prepared by BioSpyder (BioSpyder Technologies, Inc., Carlsbad, CA, United States) according to their protocol  guidelines. One μl of each RNA sample (500-660 ng/uL) was hybridized with the S1500+ beta detector oligo pool mix (2 μl per sample) using the following thermocycler settings: 10 min at 70°C, followed by gradual decrease to 45°C over 49 min, and ending with 45°C for 1 min. Hybridization was followed by nuclease digestion (24 μl nuclease mix addition followed by 90 min at 37°C), ligation (24 μl ligation mix addition followed by 60 min at 37°C, then heat denaturation at 80°C for 30 min).. Ten microliters of each ligation product were then transferred to a 96-well PCR amplification microplate that also contained 10 μl of PCR mix per well. Through amplification well-specific, “barcoded” primer pairs were introduced to templates. Five microliters of the PCR amplification products from each well were then pooled into a single sequencing library. The TempO-Seq library was then processed with a PCR clean-up kit (Machery-Nagel, Mountain View, CA, United States) prior to sequencing.