GSM6816333: 24 hours_1; Homo sapiens; RNA-Seq
Total RNA was purified using mRNeasy kit (Qiagen) and eluted in 50 uL of EB (Qiagen) polyadenylated mRNAs were enriched using Oligo dT selection, followed by cDNA synthesis using random N6-primed reverse transcription and a second-strand cDNA synthesis with dUTP instead of dTTP. The synthesised cDNA was then subjected to end-repair and 3' adenylated, followed by adaptor ligation to both ends. The dUTP-marked strand was selectively degraded using Uracil-DNA-Glycosylase (UDG). The remaining strand was amplified to generate a cDNA library suitable for sequencing prior to several cyclyes of PCR amplifcation. The PCR products were heat-denatured and the single strand DNA was cyclised by splint oligos and DNA ligase, followed by DNA nanoball synthesis and sequencing on DNBSEQ platform (BGI)