Extraction of Small RNA from Hemocytes of VPAHPND-Challenged Shrimps

The hemolymph (500 µL) of VP_AHPND-challenged shrimps at 0 and 6 h post-infection (hpi) was drawn out from the ventral sinus using a sterile syringe pre-loaded with an equal volume of anticoagulant (27 mM sodium citrate, 336 mM sodium chloride, 115 mM glucose, and 9 mM EDTA, pH 5.6)^{39 (link)}. Hemocytes were then collected by centrifugation at 800×g for 10 min at 4 °C. The hemocytes from 30 individuals were pooled and extracted for total small (s)RNA using a mirVana miRNA Isolation Kit (Ambion, Life Technologies) following the manufacturer's protocol. The total sRNA quality was evaluated on Agilent 2100 Bioanalyzer using a Small RNA Kit (Agilent). The RNA concentrations were determined by Qubit RNA HS Assay Kit on the Qubit 2.0 flourometer (ThermoFisher Scientific).