RNA-Seq of Eriocheir sinensis: eyestalks in day and night
Total RNA was extracted using the Trizol Kit (Invitrogen, USA), following manufacturer protocol. Quality and quantity were examined using 1% TAE agarose gel electrophoresis. Equal quantities (0.5 g) of RNA from five controls and five infected shrimps were separately pooled to eliminate sample variation, resulting in two samples for RNA-seq library construction with the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA), following manufacturer protocol. Library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, USA) to select cDNA. cDNA (150200 bp) were pooled to form a 180480 bp adaptor-ligated sample, to which 3 L of USER Enzyme (NEB, USA) was added; the mixture was incubated at 37 C for 15 min, then at 95 C for 5 min. Next, PCR was performed using Phusion High-Fidelity DNA polymerase plus GC buffer and 2 M betaine, along with universal PCR primers and Index (X) Primer. Amplicons were then purified using AMPure XP, and library quality was assessed with Agilent Bioanalyzer 2100. Mixed DNA libraries were diluted to 45 pM for sequencing on the Illumina Hiseq 4000TM platform.