Fragment analyzer for size profiling

RNA of each larvae (n=10 per condition) used in these experiments was extracted using NucleoSpin RNA extraction kit (Macherey-Nagel, Düren, Germany).RNA-Seq libraries were generated from 2000 ng of total RNA using the Illumina Stranded mRNA Prep mRNA Sample Preparation Kit with UDI indices (Illumina, USA) according to the manufacturer’s instructions. Surplus PCR primers were removed using AMPure XP (Beckman Coulter Life Sciences, USA). Final cDNA libraries were checked for quality and quantified using Qubit (ThermoFisher Scientific, USA) and Fragment Analyzer for size profiling (Agilent, USA), and concentration normalized using KAPA Library Quantification Kit for Illumina Platforms (Roche, USA). Sequencing was performed on an Illumina NextSeq2000 for paired-end 150 base format. Libraries were loaded in the P2 flow cell at 645 nM. The fastQ files were generated and demultiplexed using bcl2fastq v2.20 pipeline. RNA-Seq library preparation and sequencing were performed by the High Throughput Genomics Core of Biodiversity Research Center in Academia Sinica in Taipei, Taiwan (http://ngs.biodiv.tw/NGSCore/contact-location/).