85-10
Briefly, mRNA was purified from total RNA using polyT oligo-attached magnetic beads. Fragmentation was performed using divalent cations under an elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5). 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. The remaining overhangs were converted into blunt ends via exonuclease/polymerase activity. After adenylation of the 3 ends of the DNA fragments, NEBNext Adaptor with hairpin loop structure was ligated to prepare for hybridization. To select cDNA fragments preferentially 150200 bp in length, the library fragments were purified with an AMPure XP system (Beckman Coulter, Beverly, USA). Then, 3 L of USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 C for 15 min, followed by 5 min at 95 C before PCR. Then, PCR was performed with Phusion High-Fidelity DNA polymerase, universal PCR primers, and Index (X) Primer. Finally, the PCR products were purified with the AMPure XP system and the library quality was assessed using the Agilent Bioanalyzer 2100 system. The cDNA library was sequenced using Illumina HiSeqTM. After removing reads containing adapter, reads containing ploy-N and low quality reads from raw data by Illumina Casava 1.8, the clean reads were de novo assembled into unigenes using the Trinity v2.2.0 with min_kmer_cov set to 2 as the default and all other parameters set to the default