Sv total rna extraction system

Adult female 0- to 3-day-old D. sechellia 14021-0428.25 flies were fed control food (Carolina Biological Supply) or control food mixed with 1 g rotten M. citrifolia fruit pulp for 24 h. The Morinda fruit used in this experiment was harvested when fully ripe (white/gray in color and soft) from plants grown on site. The fruit was then aged in 1 L plastic containers with holes in the lid to allow air movement for 7 days at 25°C and 70% relative humidity. Seeds were removed and pulp homogenized before incorporation in food media. After treatment, flies were snap frozen in liquid nitrogen and stored at −80°C until RNA extraction. Three replicates were analyzed per treatment, with 10 flies per replicate, generating 3 control and 3 noni fed samples. RNA was extracted using the Promega SV total RNA extraction system with modified protocol (Promega; Coolon et al. 2013 (link)). RNA quality was determined using gel electrophoresis (Thermo Fisher Scientific, USA) and NanoDrop spectrophotometer (Thermo Fisher ScientificUSA). RNA was sent to the University of Michigan Sequencing Core Facility where mRNA selection was performed from total RNA using poly(A) selection. cDNA libraries were then sequenced using the Illumina Hiseq 4000 platform.

After exposure to the control or L-DOPA food sources, flies of each species were flash frozen in liquid nitrogen and kept at -80° until RNA extraction. The Promega SV total RNA extraction system with modified protocol (Coolon et al. 2012 (link)) was used to extract RNA from a homogenate of 10 whole adult female flies per replicate per species per treatment. Three biological replicates were analyzed for each species and exposure environment for a total of 18 sequencing libraries (Table 1). Prior to library preparation, NanoDrop and subsequent gel electrophoresis were used to determine the quantity and quality of RNA extracted. All RNA samples were sent to the University of Michigan Medical School DNA Sequencing Core Facility for mRNA selected library preparation and sequencing. Bar-coded sequencing libraries were made using TruSeq library preparation kits and pooled for sequencing. Uniform library representation of each library was confirmed with qPCR prior to sequencing. The pooled barcoded libraries were sequenced on two lanes of an Illumina HiSeq-4000 generating single end sequence reads for subsequent analyses.

Drosophila sechellia (14021‐0428.25) flies were reared on cornmeal medium using a 16:8 light:dark cycle at 20°C. Wandering F₁ larvae (stage L3) were collected and exposed to either control food or food containing 0.2% OA for an exposure period of 1 hr. Following exposure, larvae were snap‐frozen in liquid nitrogen and stored at −80°C until RNA extraction. RNA was extracted from a homogenate of 10 whole larvae using the Promega SV total RNA extraction system with modified protocol (Promega; Coolon, Webb, & Wittkopp, 2013). Three biological replicates were produced for each treatment for a total of six sequencing libraries. Five microliters of RNA from each extraction was checked via gel electrophoresis to confirm successful RNA extraction. RNA quality control (BioAnalyzer and NanoDrop), library preparation (TruSeq mRNA library preparation kit), and RNA sequencing (Illumina HiSeq‐4000, H4K Single End 50 Cycle) were performed by the University of Michigan Medical School DNA Sequencing Core.