title: dsU16-injected Hyposoter didymator calyx cells at pupal stage 3
organism: Hyposoter didymator
description: Calyx cells dissected from dsU16-injected Hyposoter didymator females at pupal stage 3  for pupae with pigmented thorax and abdomen (~5 day-old pupae), using three biological replicas (each corresponding to 30 to 50 calyx). The gene-specific double-stranded RNA (dsRNA) used for RNAi experiments were prepared with the T7 RiboMAX Express RNAi System (Promega) according to the manufacturers instructions. Briefly, a 600-750 bp fragment corresponding to U16 sequence was cloned into the double T7 vector L4440. Then, a transcription in vitro template DNA was PCR amplified with T7 primer and used to synthetized sense and antisense RNA strands with T7 RNA polymerase at 37C during 5 h.  After annealing and DNase treatment with the TURBO DNA-free Kit (Life Technologies), purified dsRNA were re-suspended in nuclease-free water, quantified using a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific) and examined by agarose gel electrophoresis to ensure their integrity. Injections were performed in less than one-day old female pupae using a microinjector Fentojet Express (Eppendorf) and a micromanipulator (Narishige). Approximately 0.3-0.6 ul of 2-4 ug/ul dsRNA was injected into each individual. Treated pupae were kept in an incubator until adult emergence, which occurred approximately 5 days after injection.