Instrument: NextSeq 500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Total RNA was extracted from 15 midguts (= 1 sample) using TRIzol reagent (Invitrogen, Carlsbad, CA). RNA samples were DNase-treated to remove potential contamination from genomic DNA. RNA purity and concentration were analyzed with a NanoDrop 1000 v 1.3.2 (Thermo Scientific, Wilmington, DE). The absence of RNA degradation was initially assessed by electrophoresis of 1 μg of RNA on a 1.0% agarose gel. Thereafter, the quality of each RNA sample was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) with an RNA Nano Chip. mRNA samples were further prepared for RNA-Seq analysis using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA). First-strand cDNA synthesis produced single-stranded DNA copies from the fragmented RNA by reverse transcription. After second-strand cDNA synthesis, the double-stranded DNA underwent end-repair and the 3’ends were adenylated. Finally, universal adapters were ligated to the cDNA fragments and PCR was performed to produce the final sequencing library. These template molecules were used for cluster generation and sequencing on the Illumina NextSeq 500 instrument with multiple lanes and paired-end read (2 x 75 bases). Each treatment was analyzed as three independent biological replicates.