title: GSM5700085: MSC3_1; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was isolated from cells using Qiagen RNeasy Plus Mini kit (Cat. #74134;GmbH) as per manufacturer protocol. RNA was extracted from cells using the RNeasy Mini kit (Cat#74104, Qiagen). Library preparation strategy (BGISEQ-500, RRID:SCR_017979) was adopted and performed by BGI, China. Briefly, mRNA molecules were purified from total RNA using oligo(dT) attached magnetic beads and fragmented into small pieces using fragmentation reagent after reaction a certain period at proper temperature. 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. This process amplified the cDNA fragments with adapters from previous step. PCR products were purified with Ampure XP Beads (AGENCOURT), and dissolved in EB solution. Library was validated on the Agilent Technologies 2100 bioanalyzer (2100 Bioanalyzer Instrument, RRID:SCR_018043). The double stranded PCR products were heat denatured and circularized by the splint oligo sequence. The single strand circle DNA (ssCir DNA) were formatted as the final library. The 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 100) bases reads were generated in the way of sequenced by synthesis.