title: GSM9300707: H0699; Mus musculus; RNA-Seq
library_construction_protocol: Tissue collection started at approximately 9 am with the anesthetic Avertin used via intraperitoneal injection of 0.2 mL per 10 g of body weight. Animals were perfused with ice-cold phosphate-buffered saline. The liver was the first organ harvested. The gall bladder was removed, the liver weighed, and then immediately frozen in liquid nitrogen in 20 mL scintillator vials. Roughly 20 mg of pre-pulverized liver tissue was mixed with 1 mL of TRIzol reagent at 4°C for RNA extraction. The sample was then further homogenized in TRIzol with a metallic bead for 2x30s at 25 Hz. The homogenate was transferred to a new tube (without the bead) and 200 µL of chloroform was added and mixed. Samples were centrifuged at 12,000g for 15 minutes at 4°C. ~400 µL of the clear top phase was taken and added to a tube with 400 µL isopropanol, followed by vortexing and the same centrifugation. The RNA pellet was observed and the liquid was discarded. The RNA pellet was resuspended in ethanol by pipetting, then the samples were centrifuged again. The ethanol was removed and the sample was air dried and quantified by Nanodrop. Next, samples were cleaned up using the RNEasy MinElute kits (Qiagen) as per manufacturing instructions. RNA-seq and RNA integrity (RIN) checks were performed by an Agilent 2100 Bioanalyzer, and samples with RIN ≥ 6 were retained for RNA-seq, which was run on a NovoGene HiSeq 3000 with 150 bp end paired reads after polyA+ enrichment at the University of Tennessee Health Science Center transcriptomics platform. Samples were measured with an average of 24±2.7 million reads (mean±standard deviation).