title: RNA-Seq of Zophobas morio whole gut
design: The total RNA was isolated from both the whole gut tissues using TRIzol method. In order to remove any genomic DNA contamination, the extracted RNA was treated with DNase I [New England BioLabs (NEB), USA] using manufacturers protocol. The RNA extracted using TRIzol method was checked for quantity using RNA HS assay kit (ThermoFisher Scientific #Q32851) on Qubit Fluorometer (ThermoFisher Scientific #Q33238) following manufacturers protocol. In order to measure the purity of the extracted RNA, the concentration of both the samples (fat body and whole gut) was checked using Nanodrop 1000. Finally, RNA integrity number for the samples was obtained using Tapestation 4150 with HS RNA screen tape. Ribo-Zero Plus rRNA depletion kit was used to remove abundant RNA by means of enzymatic depletion. The remaining RNA was then converted into sequencing libraries using TruSeq Stranded Total RNA reagents for Illumina systems. All the steps were performed using the Ribo-Zero Plus rRNA Depletion kit. The cleaved RNA fragments primed with random hexamers were reverse transcribed into first strand cDNA. The RNA template was removed and a replacement strand was synthesized to generate double-stranded cDNA and it was purified. In order to prevent from self-ligation, during adapter ligation reaction, one adenine nucleotide was added to the 3 ends of the blunt fragments. Multiple indexing adapters were ligated to the ends of the double-stranded cDNA fragments which in turn prepared for a hybridisation onto a flow cell. To selectively amplify, DNA molecules that were provided with adaptor molecules on both the ends, PCR was performed using PPC (PCR Primer Cocktail) that can anneal the ends of the adapters. The concentration and quality of the final libraries were checked. 1 l of the pooled library was analysed on the Agilent 2100 bioanalyzer using a DNA 1000 kit. For further quantification, 2 l library was analysed using the Qubit ds DNA BR assay kit. Libraries were diluted to the starting concentration for the sequencing system. Following dilution, libraries ready to be denatured were diluted to the final loading concentration. Paired end run of 76 cycles per read and 8 cycles per index read was set. The volumes of RSB and library needed to dilute to the starting concentration was calculated using the molarity value. Each library was diluted and 10 l of each diluted library was added to a tube for creating a pool for the sequencing system.