title: GSM9533245: U25_siLuc_Rep2; Drosophila melanogaster; ncRNA-Seq
library_construction_protocol: 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 and facilitate library multiplexing, adaptors were designed with random nucleotides placed adjacent to the ligating end. The 3′ adaptor carried six random bases at its 5′ terminus and contained sample barcodes (X) for multiplexing (5rApp/NNNNNNXXXX–Adaptor–/3ddC). The 5′ adaptor was an RNA oligo bearing four random nucleotides at its 3′ terminus (…UCUNNNN). After 3′ adaptor ligation with T4 RNA ligase 2, truncated KQ, samples were pooled as necessary, 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′ adaptor 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. 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, multiplexed and sequenced (SE50, Illumina NovaSeq6000).