GSM6701383: Control 4; Homo sapiens; RNA-Seq
Total RNA was extracted from the tissues using Trizol (Invitrogen, Carlsbad, CA, USA) according   to manual instruction. About 60 mg of tissues were ground into powder by liquid nitrogen in a 2   mL tube, followed by being homogenized for 2 minutes and rested horizontally for 5 minutes. The   mix was centrifuged for 5 minutes at 12,000×g at 4°C, then the supernatant was transferred into a   new EP tube with 0.3 mL chloroform/isoamyl alcohol (24:1). The mix was shacked vigorously for   15s, 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 equal volume of   supernatant of isopropyl alcohol, 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, then the mix   was centrifuged at 13,600 rpm for 3 minutes at 4°C to collect residual ethanol, followed by the   pellet air dry for 5-10 minutes in the biosafety cabinet. Finally, 25µL~100µL of DEPC-treated   water was added to dissolve the RNA. Subsequently, total RNA was qualified and quantified using   a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA). Oligo(dT)-attached magnetic beads were used to purified mRNA. Purified mRNA was fragmented   into small pieces with fragment buffer 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 by incubating to end   repair. The cDNA fragments obtained from 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 previous step were heated denatured and circularized by the splint oligo sequence to get the   final library. The single strand circle DNA (ssCir DNA) was formatted as the final library. The   final library was amplified with phi29 to make DNA nanoball (DNB) which had more than 300   copies of one molecular, DNBs were loaded into the patterned nanoarray and single end 50 bases   reads were generated on BGIseq500 platform (BGI-Shenzhen, China).