Sperm small RNA after Piwi and Aub knock-out in drosophila melanogaster

Small RNA sequencing was performed on drosophila sperm from wild type, as well as Piwi and Aub heterozygote knock-out flies. In addition, three parts of the drosophila testis from wild-type flies were also prepared. Testes with seminal vesicles (mature sperm) were dissected from male flies in TC-100 insect medium (Sigma). Seminal vesicles were separated from the rest of the testis. Testes were further separated into three parts stored in insect medium containing tubes. Mature sperm was extracted from seminal vesicles by puncturing the sack and spooling the sperm onto clean forceps, which transferred the sperm into insect medium containing tubes. After collection, samples were pelleted, the supernatant was discarded, and then samples were re-suspended in 250ul of Trizol (Invitrogen). All samples were snap frozen in liquid nitrogen after a maximum of 30 min of dissection.