GSM7867476: RNA-Seq of sicontrol-NS-3; Homo sapiens; RNA-Seq
Total RNA from cultured MSCs was isolated using RNA-iso (Takara, Otsu, Japan) and quantified by spectrometry at 260 and 280 nm. 1) mRNA enrichment and purification:  Oligo dT Selection (mRNA enrichment): Use oligo dT beads to enrich mRNA with poly A tail.  2) RNA fragment and reverse transcription  Fragment the RNA and first-strand cDNA was generated using random N6-primed reverse transcription, followed by a second-strand cDNA synthesis with dUTP instead of dTTP.  3) End repair, add A and adaptor ligation  The synthesized cDNA was subjected to end-repair and then was 3' adenylated. Adaptors were ligated to the ends of these 3' adenylated cDNA fragments;  4) PCR amplification  Prior to PCR amplification, the dUTP-marked strand is selectively degraded by Uracil-DNA-Glycosylase(UDG). The remaining strand is amplified to generate a cDNA library suitable for sequencing. Many rounds of PCR amplification were performed to enrich the purified cDNA template using PCR primer;  5) Single strand separation and cyclization  Denature the PCR product by heat and the single strand DNA is cyclized by splint oligo and DNA ligase;  6) DNA nanoball synthesis  7) Sequencing on DNBSEQ (DNBSEQ Technology) platform