RNA-Seq (small RNA) of Glossina morsitans: mated adult female crops and proventriculi
Bacteriomes or aposymbiotic tissues (consisting of the crop and proventriculus anterior to the bacteriome) were dissected under microscope and placed in RNAlater overnight (Invitrogen, Carlsbad, CA) at 4C. An equivalent number of tissues from females containing 1st-3rd instar larva in utero were obtained while virgin flies were age-matched. The bacteriomes or aposymbiotic tissues of 20 tsetse females were pooled for one biological sample. The bacteriomes and aposymbiotic tissue were homogenized using mechanical pestles, and total RNA was isolated using the PureLink RNA Mini Kit (Ambion, Austin, TX) following the Purifying RNA from Animal Tissues protocol and subsequently treated with Turbo DNAse I (Invitrogen). RNA quality was determined with an Agilent Bioanalyzer 2100 before sequencing. Small RNA libraries were built using NextFlex V3 Kit from BiooScientific (PerkinElmer, Waltham, MA) starting with 100ng total RNA, 1/3rd adapter dilution, and 18 cycles of PCR. Samples were sequenced on an Illumina HiSeq 2500 (Marshall University Genomics Core Facility, Huntington, WV) with single end 40bp reads.