RNA-seq of Panonychus citri: fenpropathrin-susceptible strain
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 NEBNext First Strand Synthesis Reaction BufferŁ¨5XŁŠ. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse TranscriptaseŁ¨RNaseH-ŁŠ. 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 3ĄŻ ends of DNA fragments, NEBNext Adaptor with hairpin loop structurewere ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 ŚĚl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37ĄăC for 15 min followed by 5 min at 95 ĄăC before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCRproducts were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.