RNA-seq of Drosophila melanogaster: aubHN/N11 adult ovary
Briefly, total RNA was extracted from the same ovaries as above, and mRNAs were isolated using poly-T Dynabeads (Invitrogen) according to the manufacturers instructions. Isolated mRNAs were further fragmented using fragmentation buffer (Ambion), ethanol precipitated, and reverse transcribed using Superscript II (Invitrogen) and random hexamer primers. Second-strand synthesis was performed using DNA polymerase I (Promega). cDNA was purified on a MinElute column (Qiagen), repaired with End-IT DNA repair kit (Epicentre), A-tailed with Klenow enzyme (New England Biolabs), and ligated to Illumina adaptors. Ligated cDNA was gel purified with the MinElute gel purification kit (Qiagen), PCR amplified, and gel purified again to make libraries. RNA libraries were subsequently sequenced by Illumina platform.