GSM6260258: Ctrl, rep 1; Homo sapiens; RNA-Seq
Total RNAs were prepared using a NucleoSpin RNA Plus kit (TaKaRa, 740984.50) The poly-A containing mRNA molecules using poly-T oligo-attached 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. The PCR yield was quantified by Qubit and pooled samples together to make a single strand DNA circle (ssDNA circle), which gave the final library.