title: GSM8680238: OC236 EpCAM negative 2; Homo sapiens; RNA-Seq
library_construction_protocol: total RNA was extracted with Trizol® (Life Technologies, California, USA), treated with 7 Kunitz units of DNase I (Qiagen, Hilden, Germany) and further purified on RNeasy mini columns (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNA integrity was ascertained using the 2100 Bioanalyzer (Agilent, Santa Clara, CA). 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