RNA-seq of mouse RAW264.7 macrophages treated with bone-metastatic lung cancer-derived exosomes to investigate osteoclast differentiation
RNA-seq of mouse RAW264.7 macrophages treated with bone-metastatic lung cancer-derived exosomes to investigate osteoclast differentiation
Total RNA was extracted from RAW264.7 cells after 72 hours of exosome treatment using Trizol reagent (Invitrogen, #15596026). RAW264.7 murine macrophage cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were maintained at 37°C in a humidified incubator with 5% CO₂. RAW264.7 cells were treated with 10 μg/mL of exosomes (isolated from NC-LLC or BM-LLC cell culture supernatants) for 72 hours. The culture medium was refreshed every 48 hours. Total RNA was extracted from cells using Trizol reagent (Invitrogen, #15596026) according to the manufacturer's instructions. RNA quality and concentration were assessed using a NanoDrop spectrophotometer. RNA libraries for sequencing were constructed using the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB). Briefly, mRNA was enriched, fragmented, and reverse-transcribed into cDNA. After end repair and adapter ligation, libraries were amplified by PCR. The final library quality was checked using an Agilent Bioanalyzer.