GSM8188209: Vasculogenic Mimicry Control, 48 hours, replicate 1; Homo sapiens; RNA-Seq
RNA was obtained at different timepoints of Vasculogenic Mimicry formation (2, 8, 24 and 48 hours) using TriZol (Invitrogen) and the following the manufacturers protocol. Samples were sent to BGI Genomics for the RNA-seq analysis. RNA libraries were constructed by BGI Genomics following their protocol. In brief, the first step in the workflow involves purifying the poly-A containing mRNA molecules using poly-T oligoattached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then have the addition of a single 'A' base and subsequent ligation of the adapter. The products are then purified and enriched with PCR amplification. We then quantified the PCR yield by Qubit and pooled samples together to make a single strand DNA circle (ssDNA circle), which gave the final library.