Transcriptome-based approach for gene discovery and metabolic engineering of camptothecin biosynthesis in Nothopodytes nimmoniana Graham- Root
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 go through an end repair process, the addition of a single ‘A’ base, and then ligation of the adapters. The products are then purified and enriched with PCR to create the final  cDNA library. The resulting cDNA was  subjected to sequencing on Illumina HiSeq 2000  platform. Both ends of the cDNAs are sequenced, then paired-end reads are generated.