GSM9533219: TT-INPUT_10min_Rep1; Drosophila melanogaster; RNA-Seq
Total RNA was extracted using Trizol with chloroform partitioning, precipitated with isopropanol in the presence of DTT and glycogen, and resuspended in nuclease-free water. RNA quality was assessed by Fragment Analyzer, and aliquots were reserved for input (1 µg). Following TurboDNase treatment and column purification to remove residual DNA, Input RNA underwent rRNA depletion using Drosophila-specific oligos and RNase H, as previously described (ElMaghraby et al., 2019). For pulldown samples, the depletion was performed using 1/10th of the oligos and 1/5th of the enzyme concentration used for input samples. The depletion was followed by a final TurboDNase treatment and column-based cleanup. Strand-specific Illumina sequencing libraries were prepared from rRNA-depleted RNA using the NEBNext Ultra II Directional RNA Library Prep Kit. Input samples were fragmented for 15 min during library preparation, whereas pulldown samples were fragmented for 8 min to account for prior fragmentation and maintain optimal insert sizes (200–600 nt). Libraries were amplified and sequenced on an Illumina platform.