RNA-seq of Danio rerio: 25dpf wt trunk 1
The first step in the workflow involves purifying 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. We then quantified the PCR yield by Qubit and pooled samples together to make a single strand DNA circle,which gave the final library.