title: Transcriptome analysis of the rice weevil midgut at day 9 after ultimate insect molting
design: 1. Biological material  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 S. pierantonius. Aposymbiotic insects were obtained as previously described [40]. The aposymbiotic status was confirmed by PCR and histology. Weevil larvae and nymphs grow naturally inside the wheat grain. After completing metamorphosis with an ultimate insect molting (UIM, day 0 (D0) of adulthood), insects remain inside the grains three more days before emerging as adults.   Insect guts were dissected from fourth instar larvae in Buffer A (25 nM KCl, 10 nM MgCl2, 250 nM Sucrose, 35 nM Tris/HCl, pH=7.5). Tissues were pooled and stored at -80°C, prior to RNA extraction.     2. RNA isolation and libraries construction for RNAseq  RNAseq libraries were constructed from symbiotic insect guts at day 1, day 6 and day 9 after UIM (respectively D1, D6 and D9). Total RNA from 20 guts was extracted with Trizol reagent (Invitrogen) following the manufacturer’s instructions. RNA was incubated with 1 U/µg of RQ1 RNase-free DNase for 30 min, at 37°C then purified with RNeasy micro kit (QIAGEN) clean-up protocol. Two independent replicates of 1 µg of RNA sample from D1, D6 and D9 guts were provided to ProfileXpert (Lyon, France) and sequenced on an Illumina HiSeq2000 instrument following the manufacturer’s protocol and generate paired-end data (51 bp reads).