Chrysomya megacephala (Fabricius) transcriptome by Sun Yet-sen University
In brief, beads with Oligo (dT) were used to isolate poly (A) mRNA after total RNA was collected from the negative control group. Fragmentation buffer was added for cuting mRNA to short fragments. Taking these short fragments as templates, random hexamer-primer was used to synthesize the first-strand cDNA. The second-strand cDNA was synthesized using buffer, dNTPs, RNaseH and DNA polymerase I, respectively. Short fragments were purified with QiaQuick PCR extraction kit and resolved with EB buffer for end reparation and adding poly (A). After that, the short fragments were connected with sequencing adapters. And, after the agarose gel electrophoresis, the suitable fragments were selected for the PCR amplification as templates. At last, the library could be sequenced using Illumina HiSeq™ 2000.