GSM9533229: si-Yb-Rep1; Drosophila melanogaster; ncRNA-Seq
sRNAseq libraries were prepared by first purifying Argonaute–sRNA complexes with TraPR resin columns. OSCs were harvested using trypsin, washed 2x with PBS and stored at -70°C after snap freezing cell-pellets in liquid nitrogen. OSCs were lysed by adding 330 µL of TraPR lysis buffer. OSC lysates were clarified, normalized to equal protein amounts (depending on the experiment 70-120 µg total protein) after Bradford measurements, applied to TraPR columns, and Argonaute-bound RNAs were eluted, followed by phenol–chloroform extraction and precipitation. To minimize ligation bias, adapters were designed with four random nucleotides (4N) adjacent to the ligating ends. The 3′ adapter carried four random bases at its 5′ terminus (5rApp/NNNN–Adaptor–/3ddC), and the 5′ RNA adapter contained four random nucleotides at its 3′ terminus (…UCUNNNN). After 3′ adapter ligation with T4 RNA ligase 2, truncated KQ, samples were spiked with fluorescently labeled and ligation-blocked oligos as size controls and gel-purified on a 12.5% polyacrylamide–urea gel. Small RNAs were isolated using the Zymo ZR small RNA PAGE Recovery kit and subjected to 5′ adapter ligation with T4 RNA ligase 1. Following cleanup with Zymo RCC5 columns, reverse transcription was performed. Libraries were PCR-amplified with KAPA HiFi polymerase and dual-index barcoded primers to enable multiplexing at the sequencing stage. Amplification was monitored in real time with EvaGreen, and reactions were stopped upon reaching 3,500–4,000 RFU to prevent overamplification. Final libraries were size-selected on low-melt agarose gels, gel-purified (Zymo), quantified with Qubit, and sequenced (SE50, Illumina NextSeq 2000).