title: RNA sequencing of two Hemarthria species using Illumina HiSeq 2500
design: In briefly, after the total RNA were extracted, mRNA in the samples was purified and enriched using magnetic oligo(dT)-rich beads and chemically fragmented to short pieces in fragmentation buffer under an elevated temperature. These cleaved mRNA fragments then were used as templates to synthesize the first-strand cDNA under the effect of reverse transcriptase and random hexamer-primers. After that, second-strand cDNA was synthesized by buffer, dNTPs, DNA polymerase I and RNaseH. After purification by Agencourt AMPure XP beads, the double-strand cDNA was treated to repair ends by T4 DNA polymerase and Klenow DNA polymerase, added a single A base and ligated to sequencing adapters, and then selected suitable length fragments for downstream analysis as sequencing templates by Agencourt AMPure XP beads. Then, PCR amplification was performed using Phusion High-Fidelity DNA polymerase to enrich the purified cDNA template. After cDNA library was built, in order to ensure the quality of the library, the concentration and insert size of the library were detected respectively by Qubit® 2.0 Fluorometer and Agilent 2100 Bioanalyzer, and the effective concentration of the library was accurately quantified with a Q-PCR method. Finally, the qualified cDNA library was sequenced on Illumina HiSeq™2500 sequencing platform using paired-end technology in a single run.