GSM5708360: MCF7-Ctrl RNA-Seq; Homo sapiens; RNA-Seq
Total RNA samples were extracted from both MCF7-TWIST1 and MCF7-Ctrl using the TRIzol reagent (15596018; Thermo Fisher) followed by purification with the RNeasy Mini Kit (74106; Qiagen) according to manufacturer's instructions. Quality of the samples were checked by using the NanoDrop spectrophotometer and Agilent Bioanalyzer 2100. 250 ng of total RNA samples (measured by picogreen) that had passed the quality check was used for creating a double-stranded DNA library. ERCC RNA Spike-In Controls were added to each sample according to the manufacturer's protocol. The fragmented and 3' poly (A)-selected portion of total RNA together with random primers were used to create the cDNA. In the process of second strand synthesis, dTTP was replaced with dUTP to quench the second strand during amplification amd thereby achieve strand specificity. The created cDNA fragments were blunt ended, attached with an adenosine to the 3' end, and finally ligated with unique adapters to the end. The ligated products were amplified with 15 PCR cycles. The resulting libraries were quantitated using the NanoDrop Spectrophotometer and the fragment sizes were assessed with the Agilent Bioanalyzer. To determine the concentration of adapter-ligated fragments, the libraries were further quantitated by qPCR by using the Applied Biosystems ViiA7 Real-Time PCR System and a KAPA Library Quant Kit.