Iso-Seq of whole Drosophila affinis males
I sorted male and female flies, made simple by the distinctive red coloring of affinis group male testes, visible through the abdomen. Approximately 40 male and 40 female flies, ages 3-5 days, were anesthetized with CO2, frozen on dry ice, stored at -72C, and mechanically homogenized with a pestle in Zymo DNA/RNAShield. RNA was extracted using the ZymoBIOMICS Quick-RNA miniprep kit (Zymo Research, R1655) and eluted into 30 uL. Iso-seq libraries were created according to the Preparing Iso-Seq v2 libraries protocol from PacBio, with barcoded cDNA primers for multiplexing with three other Drosophila samples. Libraries were pooled, bound, and cleaned according to the Revio SPRQ Polymerase kit. Libraries were then loaded onto the Revio instrument and run using the application presets.