title: Sample 29
organism: Drosophila melanogaster
description: Protocols: Dissection into RNAlater solution Dissected 6h into the light cycle into ice-cold RNAlater. The base (1SY) medium contained 100 g/l autolysed yeast (MP Biomedicals, OH, USA), 50 g/l sucrose (Tate & Lyle, London, UK), 15 g/l agar (Sigma-Aldrich, Dorset, UK), 30 ml/l nipagin (Chemlink Specialities, Dorset, UK), and 3 ml/l propionic acid (Sigma-Aldrich, Dorset, UK). The EAA-replete control medium comprised DR food with an EAA supplement dissolved in pH 4.5 water (final concentrations in fly media: L-arginine 0.43 g/l, L-histidine 0.21 g/l, L-isoleucine 0.34 g/l, L-leucine 0.48 g/l, L-lysine 0.52 g/l, L-methionine 0.1 g/l, L-phenylalanine 0.26 g/l, L-threonine 0.37 g/l, L-tryptophan 0.09 g/l, L-valine 0.4 g/l: all suppled by Sigma). EAA+rapamycin food consisted of EAA-replete medium with a rapamycin supplement (LC laboratories, MA, USA) dissolved in ethanol, to a final concentration of 200 µM in the diet. Outbred wild-type Dahomey flies bearing the endosymbiont Wolbachia were cultured on a 12:12 light cycle at 25oC and 60% humidity, on 1SY medium. All flies were maintained at large population sizes to maintain outbred genetic diversity. Eggs were collected following an 18h lay on grape juice agar, added to bottles of 1SY at a standardised density and cultured to adulthood. Newly emerged experimental females were mated on fresh food for 48h, lightly CO2 anaesthetised, then allocated to experimental diets without males (10 females/vial). Qiagen total RNA isolation kit Illumina TruSeq Stranded mRNA Sample Prep kit