RNA-Seq of Glossina brevipalpis midgut male teneral
Bacteriomes and midguts (flanked by the bacteriome and the Malpighian tubules) were microscopically dissected and placed in RNAlater (Invitrogen, Carlsbad, CA) at -20C. The bacteriomes or intestinal tracts of 20 teneral tsetse of each sex were pooled for one biological sample. Bacteriomes and guts were homogenized and total RNA was extracted using a MasterPure RNA purification kit (Epicentre, Madison, WI) according to the manufacturers protocol for tissue samples. DNA was removed from the RNA samples using a Turbo DNA-free kit (Ambion, Austin, TX) following the rigorous DNase treatment option. The RNA concentration was measured using a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA) and an Agilent 2000 Bioanalyzer RNA Nano chip was used to validate RNA sample quality and integrity. mRNA Library preparation was performed at the WVU Genomics Core Facility by using 1 ug of total RNA and Ribo-Zero Gold Epidemiology kit (Illumina, San Diego, CA) following the manufacturers recommended protocol. Following cDNA synthesis, libraries were quantified via Qubit fluorometer with high sensitivity DNA reagent and run on an Agilent high sensitivity DNA chip to determine average library size. The libraries were pooled in equimolar amounts and sequenced using the Illumina HiSeq 1500 platform (2 by 51 bp) at Marshall University.