title: GSM4774845: A2: aged sample 002; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was extracted from the tissues using Trizol (Invitrogen, Carlsbad, CA, USA) according to the instruction manual. About 60 mg of tissues were ground in liquid nitrogen in a 2 mL tube. After homogenization for 2 min, samples were rested horizontally for 5 min. The mix was centrifuged for 5 min at 12,000×g at 4°C, and then the supernatant was transferred into a new tube with 0.3 mL chloroform/isoamyl alcohol (24:1). The mix was shaken vigorously for 15 sec, and then centrifuged at 12,000×g for 10 min at 4°C. After centrifugation, the upper aqueous phase was transferred into a new tube and an equal volume of isopropyl alcohol was added, then centrifuged at 13,600 rpm for 20 min at 4°C. After removing the supernatant, the RNA pellet was washed twice with 1 mL 75% ethanol, and then the mix was centrifuged at 13,600 rpm for 3 min at 4°C to collect residual ethanol, followed by air drying for 5-10 min. Subsequently, RNA was resuspended in 25~100 μL DEPC water, and then qualified using a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA). Oligo (dT)-attached magnetic beads were used to purify mRNA. Following purification, the mRNA was fragmented into small pieces using divalent cations at appropriate temperature. Then first-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. Afterwards, A-Tailing Mix and RNA Index Adapters were added and incubated to end repair. The cDNA fragments obtained from the previous step were amplified by PCR, and products were purified by Ampure XP Beads, then dissolved in EB solution. The product was validated on the Agilent Technologies 2100 bioanalyzer for quality control. The double stranded PCR products from the previous step were heated, denatured, and circularized by the splint oligo sequence to obtain the final library. The single strand circle DNA (ssCir DNA) was formatted as the final library.