RNA-Seq of PK-15 cells
Oligo dT Selection (mRNA enrichment): Use oligo dT beads to enrich mRNA with poly A tail.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.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;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;Denature the PCR product by heat and the single strand DNA is cyclized by splint oligo and DNA ligase; DNA nanoball synthesisSequencing on DNBSEQ (DNBSEQ Technology) platform