Qubit apparatus

Fecund adult females, cultured as described in Section 2.1, were used to prepare RNA for the transcriptomic analysis. Total RNA was isolated from the two biological replicates of five worms each (two wells each of treated and untreated parasites) after 2 days culture with and without 20E. The worms were flash frozen in liquid nitrogen. RNA was extracted from the worms using Trizol LS (Invitrogen, Carlsbad, CA, USA) (Griffiths et al., 2009 (link); Choi et al., 2011 (link)). The biological replicates of the 20E-treated and untreated samples were lysed individually using TissueLyse II (Qiagen, Valencia, CA, USA) followed by chloroform extraction, isopropanol precipitation and elution in 0.1×TE buffer (1 mM Tris-HCl, pH 8.0, 0.1 mM EDTA). The samples were treated with DNase I (Ambion, Austin, TX, USA) according to the manufacturer's instructions. The RNAs were subjected to drop dialysis using 45 nm Millipore membranes (EMD Millipore, Billerica, MA, USA) against 0.1×TE buffer at 4°C for 2 – 4 h and the RNA was then collected from the membranes. Purity of the samples was assessed using a NanoDrop apparatus (Thermo Scientific, Waltham, MA, USA). The quantity of RNA was determined using a Qubit apparatus (Thermo Fisher, Carlsbad, CA, USA). The purified RNA was stored at −80°C.