miRNA-seq of exosomes derived from bone-metastatic lung cancer cells
Exosomes were collected and purified from the cell culture supernatant of NC-LLC and BM-LLC cells via differential ultracentrifugation. Lewis lung carcinoma (LLC) cells (NC-LLC and BM-LLC) were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% exosome-depleted fetal bovine serum (FBS) at 37°C in a 5% CO₂ atmosphere. Total RNA, including small RNAs, was extracted from the purified exosomes using the miRNeasy Serum/Plasma Kit (Qiagen) according to the manufacturer's instructions. Small RNA libraries were constructed using the NEBNext Multiplex Small RNA Library Prep Kit for Illumina. Briefly, small RNA molecules were ligated to 3' and 5' adapters, reverse transcribed, and amplified by PCR. The final cDNA libraries were size-selected to enrich for fragments containing microRNAs.