GSM8765802: CTRU2001CE.08, Healthy, D7; Homo sapiens; RNA-Seq
Oligo (dT)-attached magnetic beads were used to purify 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. A second-strand cDNA synthesis is followed and dUTP was used instead of dTTP. Afterwards, the purified double-stranded cDNA was End Repaired, A-Tailed, and adapter ligated. The cDNA fragments obtained from previous step were digested with UDG enzyme to remove the U-labeled second-strand template and were amplified by PCR.  The final products were purified by Ampure XP Beads, then dissolved in EB solution. The product was validated on the Agilent Technologies 2100 bioanalyzer DNA analysis for quality control. The double-stranded PCR products from previous step were heat 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 (in-lane control) together to make DNA nanoball (DNB) which had more than 300 copies of one molecular, DNBs were loaded into the patterned nanoarray, and pair-end 100 bases reads were generated on BGI-G400 platform.