Stranded mrna seq kit

Total RNA was extracted from midgut tissue using Qiagen RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) and treated with RNase Free DNase (Qiagen, Valencia, CA, USA) as per manufacturer’s protocol. RNA quality was assessed with an Agilent 2100 Bioanalyzer and a Eukaryote Total RNA Nano Series II chip (Agilent, Santa Clara, CA, USA). RNA ribosomal integrity numbers (RIN) from all samples except one, ranged between 8.1 and 9.1, where any RIN above eight is considered high quality (Fleige & Pfaffl, 2006 (link)). RNA was quantified with Quant-iT™ RiboGreen® reagent and a Labsystems Fluoroskan Ascent™ Microplate Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Samples were precipitated with 1/10^th volume sodium acetate and 2.5x volume pure ethanol and stored at −80 °C prior to shipment. Eight samples with RIN greater than eight from each treatment were shipped to the Georgia Genomic Facility (GGF) for library preparation and sequencing. The quality of the total RNA was confirmed by GGF using an Agilent 2100 Bioanalyzer. GGF prepared barcoded cDNA libraries using a KAPA Stranded mRNA-Seq Kit (Wilmington, MA) and sequenced them on the Illumina NextSeq 500 using paired-end sequencing with a NextSeq 2×75 High Output Flow Cell.