title: GSM9533218: TT-SLAMseq_10min_Rep2; Drosophila melanogaster; RNA-Seq
library_construction_protocol: 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) and pulldown (50 µg) fractions. For pulldown samples, total RNA was fragmented to 200–600 nt by heating at 94 °C for 60 s in fragmentation buffer, followed by precipitation with sodium acetate and ethanol. 4sU-labeled transcripts were selectively biotinylated using MTSEA-biotin-XX (MTS-biotin) in a freshly prepared 10× biotinylation buffer (20 mM HEPES pH 7.8, 1 mM EDTA) for 2 h at 25 °C with rotation, protected from light. Unreacted biotin was removed by acid phenol–chloroform extraction, and biotinylated RNA was recovered by ethanol precipitation. Biotinylated transcripts were enriched using µMACS streptavidin magnetic beads (Duffy et al., 2015). Denatured RNA (65°C, 10 min, snap cool on ice) was incubated with streptavidin beads for 15 min at room temperature, loaded onto pre-equilibrated µMACS columns, washed three times with high-salt buffer (100 mM Tris-HCl pH 7.5, 10 mM EDTA, 1 M NaCl, 0.1% Tween-20), and eluted twice with 100 mM DTT. Eluted RNA was purified using RNeasy MinElute columns and concentrated to 15 µL. To introduce 4sU-dependent mismatch signatures for SLAM-seq identification, both input and pulldown fractions were subjected to iodoacetamide (IAA) alkylation. RNA was incubated with 10 mM IAA in 50 mM sodium phosphate buffer (pH 8.0) and 50% DMSO at 50 °C for 15 min; the reaction was then immediately quenched with DTT and the RNA was ethanol precipitated. Following TurboDNase treatment and column purification to remove residual DNA, both fractions 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.