GSM5525105: FRT19A Control; Drosophila melanogaster; RNA-Seq
Around 50 guts were isolated in cold RNAase free water. Total RNA was extracted from each sample using Tri-reagents (Sigma). The quality of RNA was measured in Agilant bio-analyzer. The qulified RNA of each sample send to BGI for making library preparation and sequencing on BGISeq-500 platform. The first step 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 and  quantified the PCR yield by Qubit and pooled samples together to make a single strand DNA circle (ssDNA circle), which gave the final library.