GSM9067520: TC-1 tumor, circRNA vaccine, Day12, rep4; Mus musculus; RNA-Seq
Frozen tumor tissues were homogenized in 1 ml of Trizol reagent. RNA was extracted with trizol-chloroform and preciptated by isopropanol. 1 μg total RNA was used for the following library preparation. For mRNA enrichment, poly(A) mRNA isolation was performed using Oligo(dT) beads. The mRNA fragmentation was performed using divalent cations and high temperature. Priming was performed using Random Primers. First strand cDNA synthesis was carried out using reverse transcriptase, followed by second-strand cDNA synthesis. The purified double-stranded cDNA was then treated to repair both ends and add a dA-tail in a single reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of adaptor-ligated DNA was then performed using DNA Clean Beads. Each sample was then amplified by PCR using P5 and P7 primers, and the PCR products were validated.