GSM9960363: MCF-7 sgUBE2C pooled RNA-seq; Homo sapiens; RNA-Seq
Total RNA was independently extracted from three separately cultured cell preparations for each condition using TRIzol reagent (Invitrogen, catalog no. 10296028) according to the manufacturer's instructions. The three RNA preparations from each condition were pooled after RNA extraction and before library preparation to generate one pooled RNA-seq sample per condition. For the sgCtrl and sgUBE2C libraries, mRNA-sequencing libraries were prepared using the TruSeq Stranded mRNA Library Prep Kit with poly(A) selection according to the manufacturer's instructions. For the Vector and UBE2C-OE libraries, polyadenylated mRNA was purified using poly-T oligo-attached beads and fragmented prior to cDNA synthesis. First-strand cDNA was synthesized using reverse transcriptase and random primers, followed by second-strand synthesis using dUTP in place of dTTP. A single adenine nucleotide was added to the 3′ ends of the double-stranded cDNA fragments, followed by indexed adapter ligation and PCR amplification. Library quality was assessed using an Agilent 2100 Bioanalyzer and a real-time PCR system.