GSM6533721: dFOXO-null_male_totalsmall_05d_rep1; Drosophila melanogaster; RNA-Seq
RNA was extracted from 40 whole flies with Trizol reagent (Invitrogen) and sized selected to 19 to 35 nt fragments using 17% 19:1 acrylamide:bisacrylamide urea gels (National Diagnostics). Gels were sliced from 19-35 nt, crushed, and eluted overnight in 500 µl 0.3 M sodium acetate with rotation. 20 µg of glycogen and small RNA spike ins (Gainetdinov, Colpan et al. 2018) were added to the supernatant before it was precipitated with 750 µl isopropanol at -20 °C for 1 hour, centrifuged at 15,000 rpm for 30 minutes at 4 °C, washed in 80% EtOH, air dried, and resuspended in 50 µl H2O. Small RNA libraries were constructed using the NEBNext® Small RNA Library Prep Set for Illumina®(New England Biolabs).