First transcriptome and digital gene expression analysis in Neuroptera with an emphasis on chemoreception genes in Chrysopa pallens (Rambur)
Total RNA was extracted by SV Total Isolation System (Promega, Madison, WI, USA) following the manufacturer’s instructions. cDNA library construction and Illumina sequencing of the samples were performed at Beijing Genomics Institute – Shenzhen, Shenzhen, China [73]. Briefly, oligo(dT) beads were used to isolate poly (A) mRNA from 20 µg of pooled total RNA. To interrupt mRNA into short fragments, fragmentation buffer and divalent cations were used at 94 °C for 5 min. Using these short fragments as templates, random hexamer-primers were used to synthesize first-strand cDNA. Second-strand cDNA was generated using buffer, dNTPs, RNAseH, and DNA polymerase I. After end-repair and ligation of adaptors, the products were amplified by PCR and purified with QIAquick PCR extraction kit (Qiagen, Venlo, Netherlands) and resolved with EB buffer for end reparation and adding poly (A). Then, the short fragments connected to sequencing adapters and detected by agarose gel electrophoresis were selected as templates for PCR amplification and sequencing using Illumina HiSeq™ 2000 (San Diego, CA, USA).