Small RNA FISH in Drosophila Testes

smRNA-FISH was conducted as described^{61 (link)}. Testes from 2–3-day-old flies were dissected in 1× PBS, fixed in 4% formaldehyde in 1× PBS for 30 min, washed in PBS and permeabilized in 70% ethanol overnight at 4 °C. The following day, testes were rinsed with wash buffer (2× saline-sodium citrate and 10% formamide) and hybridized overnight at 37 °C in hybridization buffer (2× saline-sodium citrate, 10% dextran sulfate (Sigma, D8906), 1 mg ml⁻¹Escherichia coli tRNA (Sigma, R8759), 2 mM vanadyl ribonucleoside complex (NEB, S142), 0.5% bovine serum albumin (Ambion, AM2618) and 10% formamide). Following hybridization, samples were washed three times in wash buffer for 20 min each at 37 °C and mounted in VECTASHIELD with 4′,6-diamidino-2-phenylindole (DAPI, Vector Labs). Fluorescently labelled probes were added to the hybridization buffer to a final concentration of 100 nM. DNA oligo probes to detect Ste and Su(Ste) RNA were conjugated with Quasar 570, Cy3 or Cy5 fluorophores (Biosearch Technologies and IDT; for probe information, see Supplementary Table 5). Images were acquired using an upright Leica TCS SP8 confocal microscope with a 63× oil immersion objective lens (numerical aperture 1.4) and processed using ImageJ.