Dual RNA-seq of Sitophilus oryzae bacteriome from late nymphs
Sitophilus oryzae weevils are reared on wheat grains at 27.5 C and at 70 % relative humidity. The Bouriz strain was chosen in this work because it is free of any facultative symbionts, including Wolbachia, and it harbors only Sodalis pierantonius. Weevil larvae and pupae grow naturally inside the wheat grain. Fifteen insect guts per replicate were dissected from fourth instar larvae, early nymphs, nymphs and late nymphs in Buffer A (25 nM KCl, 10 nM MgCl2, 250 nM Sucrose, 35 nM Tris/HCl, pH = 7.5). Three biological replicates were performed. Tissues were pooled and stored at 80 C, prior to RNA extraction.Total RNA was extracted with Trizol reagent (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA) following the manufacturers instructions. RNA was incubated with 1 U/g of RQ1 RNase-free DNase (Promega, Madison, WI, USA) for 30 min, at 37 C then purified with RNeasy micro kit (Qiagen, Hilden, Germany) clean-up protocol. Total RNA concentration and quality were checked using the Qubit Fluorometer (ThermoFisher Scientific) and the Agilent 2200 TapeStation (Agilent Technologies, Santa Clara, CA, USA). Dual RNA-seq libraries were then prepared using the Ovation Universal RNA-seq System (NuGEN, San Carlos, CA, USA) following the manufacturers instructions and sequenced on an Ion Torrent Proton platform (Life Technologies, Carlsbad, CA, USA) at the sequencing platform of the IGFL (Institut de Gnomique Fonctionnelle de Lyon, Ecole Normale Suprieure de Lyon, France).