GSM6046685: Fasted MEP rep2; Mus musculus; RNA-Seq
GSM6046685
Total RNA was extracted from the tissues using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Approximately 60 mg of tissue was ground into powder in liquid nitrogen in a 2 mL tube, homogenized for 2 minutes and rested horizontally for 5 minutes. The mix was centrifuged for 5 minutes at 12,000×g at 4°C, and then the supernatant was transferred into a new EP tube with 0.3 mL chloroform/isoamyl alcohol (24:1). The mix was shaken vigorously for 15 s and then centrifuged at 12,000×g for 10 minutes at 4°C. After centrifugation, the upper aqueous phase where RNA remained was transferred into a new tube with an equal volume of isopropyl alcohol supernatant and then centrifuged at 13,600 rpm for 20 minutes at 4°C. After deserting 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 minutes at 4°C to collect residual ethanol, followed by pellet air drying for 5-10 minutes in a biosafety cabinet. Finally, 25 µL~100 µL of DEPC-treated water was added to dissolve the RNA. Subsequently, total RNA was qualified and quantified using a NanoDrop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA). Oligo(dT)-attached magnetic beads were used to purify mRNA. Purified mRNA was fragmented into small pieces with fragment buffer at the appropriate temperature. Then, first-strand cDNA was generated using random hexamer-primed reverse transcription, followed by second-strand cDNA synthesis. Afterwards, A-Tailing Mix and RNA Index Adapters were added by incubation for end repair. The cDNA fragments obtained from the previous step were amplified by PCR, and the products were purified 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 from the previous step were heated, denatured and circularized by the splint oligo sequence to obtain the final library. Single strand circle DNA (ssCir DNA) was formatted as the final library. The final library was amplified with phi29 to make DNA nanoballs (DNBs), which had more than 300 copies of one molecule. DNBs were loaded into the patterned nanoarray, and paired-end 100-base reads were generated on the BGISeq500 platform (BGI-Shenzhen, China).