BGISEQ-500 sequencing; RNA-seq of liver tissue from Macaca fascicularis with high fat high sugar treatment or with spontaneous diabetes
RNA-seq of liver tissue from Macaca fascicularis with high fat high sugar treatment or with spontaneous diabetes
The liver tissues were collected from Macaca fascicularis and frozen in liquid nitrogen immediately.The tissues were transfered to a  freezer at -80 ℃ for storage. Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instruction. In Brief, about 60 mg of tissues were ground into a powder in liquid nitrogen, and homogenized in the RNA extraction buffer for 2 min, followed by resting horizontally for 5 min. The mix was then centrifuged for 5 min with 12,000 ×g at 4°C, and the supernatant was transferred into a new tube with 0.3 mL chloroform/isoamyl alcohol (24:1). The mix was shacked vigorously for 15 s and centrifuged at 12,000 ×g for 10 min at 4°C. The upper aqueous phase was transferred into a new tube and mixed with an equal volume of isopropyl alcohol, followed by centrifugation at 13,600 rpm for 20 min at 4°C. Then the RNA pellet was washed twice with 1 ml 75% ethanol and centrifuged at 13,600 rpm for 3 min at 4°C to desert the residual ethanol. After 5-10 min of air dry in a biosafety cabinet, the RNA pellet was dissolved in 25µL~100µL of DEPC-treated water, followed by qualification and quantification using a NanoDrop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA), respectively. Oligo(dT)-attached magnetic beads were applied to purify mRNA from the total RNA. Purified mRNA was fragmented into small pieces with fragment buffer. Then the first-strand cDNA was generated using random hexamer-primed reverse transcription, and the second-strand cDNA was synthesized. A-Tailing Mix and RNA Index Adapters were then added by incubation. The cDNA fragments were amplified by PCR, followed by purification by Ampure XP Beads, and then dissolved in EB solution. The product was validated on an Agilent Technologies 2100 bioanalyzer for quality control. The double-stranded PCR products were heated to denature and circularized by the splint oligo sequence to construct the final library.