RNA-Seq of Parabotia fasciatus
Total RNA was extracted using a TRIzol Reagent Kit (Invitrogen, Life Technologies, MD). RNA degradation and contamination were monitored on 1% agarose gels. The purity, concentration and integrity of RNA samples were tested using NanoDrop 2000 and Agient2100/LabChip GX. A total amount of 1 g RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNextUltra RNA Library Prep Kit for Illumina (NEB, USA) following manufacturers recommendations. Then the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). 3 l 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. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturers instructions. After cluster generation, the library preparations were sequenced on a sequencing platform and paired-end reads were generated. The sequences were further processed with a bioinformatic pipeline tool, BMKCloud (www.biocloud.net) online platform. Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. At the same time, Q20, Q54, GC-content and sequence duplication level of the clean data were calculated. All the downstream analyses were based on clean data with high quality.