title: GSM9533292: shWhite_total-sRNA; Drosophila melanogaster; ncRNA-Seq
library_construction_protocol: Total RNA was extracted from ovariesusing TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Briefly, ovaries were lysed in TRIzol, followed by phase separation with chloroform. The aqueous phase was recovered, and RNA was precipitated with isopropanol, washed with 75% ethanol, and resuspended in nuclease-free water. RNA concentration and purity were assessed by NanoDrop spectrophotometry (A260/280 and A260/230 ratios). To enrich for small RNAs and deplete ribosomal RNA, 10 µg of total RNA was subjected to 2S rRNA depletion using a biotinylated antisense oligonucleotide (5'–Bio-AGTCTTACAACCCTCAACCATATGTAGTCCAAGCAGCACT–3') immobilized on MyOne Streptavidin C1 magnetic beads (Invitrogen). RNA was denatured at 80 °C for 2 min, hybridized to the bead-bound probe at 50 °C for 1 h with rotation, and the rRNA-depleted supernatant was recovered by magnetic separation and ethanol precipitation. The depleted RNA was resuspended in nuclease-free water and used directly for library preparation. Small RNAs were isolated by resolving the mixture on a 12.5% polyacrylamide–urea gel and excising the region flanked by the spike-in signals. These spike-ins were ligated to conventional 4N adapters alongside the endogenous small RNA population. Following each ligation step, libraries were gel-purified, with band excision guided by the radioactive signal. After reverse transcription and PCR amplification, the spike-in sequences were removed from the library via restriction digestion. The libraries  were pooled at the final stage using PCR-introduced dual or single index barcodes.