Anaesthetized adult files were pinned down and T3 legs were removed to expose internal tissues. Accessory glands (with incomplete recovery of attached seminal vesicle) were collected, with testes excluded from the sample. Tissues were frozen on dry ice.
Total RNA was isolated using Trizol, then Qiagen RNeasy spin columns. mRNA was isolated using Dynal oligo(dT) beads, then fragmented using divalent cations under elevated temperature, followed by sequential ligation of RNA linkers to the 5’ and 3’ ends. Next, reverse transcription was performed using a primer complementary to the 3’ linker and PCR was performed using primers complementary to both linkers. ~ 300 bp fragments were isolated from an agarose gel and gel-purified again.
Read length (bases):83
The samples were quantitated using a Nanodrop, and loaded onto a flow cell for cluster generation and sequenced on an Illumina Genome Analyzer II using either single read or paired end protocols (Illumina).
