GSM9321451: LUT089PS; Homo sapiens; ncRNA-Seq
Total RNA was extracted from needle biopsies using the Direct-zol RNA MicroPrep Kit (Zymo Research, Cat. #R2062) according to the manufacturer's instructions, including on-column DNase I treatment to remove genomic DNA contamination. For DOPS samples, RNA was isolated from 500 μl of solution using the same kit, following the manufacturer's protocol for liquid samples, and eluted in 15 μl of RNase-free water. Purified RNA was stored at –80 °C, and repeated freeze–thaw cycles were avoided to minimize RNA degradation. Small RNA libraries were prepared using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs, Cat. #E7330) following the manufacturer's instructions. For each sample, 100 ng of tissue RNA or 5 μl of DOPS RNA was used as input. Indexing was performed using the NEBNext Multiplex Oligos for Illumina, with PCR amplification carried out for 15 cycles for tissue RNA libraries and 18 cycles for perfusate RNA libraries. Amplified products were resolved on an 8% polyacrylamide gel (Thermo Fisher Scientific, Cat. #EC6215BOX), and target bands were excised and purified. Equal amounts of libraries were pooled and sequenced on an Illumina NextSeq 500 platform, generating approximately 10 million 75 bp single-end reads per sample.