GSM8510233: midgut,AF,rep1; Spodoptera frugiperda; RNA-Seq
Total RNA was extracted from the midgut samples using the TRIzol reagent kit (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions. Total RNA was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase(RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified. After the library construction is completed, preliminary quantification is carried out using Qubit, and the library is diluted to 1.5ng/ul. Subsequently, the inserted fragments of the library are detected. Once the inserted fragments meet expectations, the effective concentration of the library is accurately quantified using qRT PCR to ensure the quality of the library. The qualified libraries were pooled and sequenced on sequencing platforms, according to effective library concentration and data amount required.