GSM5484653: obesity_2; Homo sapiens; RNA-Seq
Total RNA was extracted from the skeletal muscle tissues using Trizol (Invitrogen, Carlsbad, CA, USA) according to manual instruction. Oligo(dT)-attached magnetic beads were used to purified mRNA. Purified mRNA was fragmented into small pieces using fragment buffer at appropriate temperature. Then First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. The synthesized cDNA was subjected to end-repair and then was 3' adenylated. Adapters were ligated to the ends of these 3' adenylated cDNA fragments. 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 double stranded PCR products were heated denatured and circularized by the splint oligo sequence to get the final library. The final library was amplified with phi29 to make DNA nanoball (DNB) which had more than 300 copies of one molecular. The DNBs were load into the patterned nanoarray and single end 50 (pair end 150) bases reads were generated on BGIseq500 platform (BGI-Shenzhen, China).