GSM8257327: cochlear explants, PBS, Control2; Mus musculus; RNA-Seq
The sEV-free FBS medium was used to culture the cochlear explants with cisplatin for 48 h. The culture supernatant was collected and stored at 4℃, and sEVs were isolated from the supernatant within one week. A series of sequential centrifugation steps were employed to remove cells and cellular debris, including 600 × g for 10 min and 2000 × g for 20 min at 4°C. To remove large vesicles, the supernatant was then filtered through a 0.22 μm filter (Millipore, SLGPR33RB) and centrifugated at 16500 × g at 4°C for 20 min. After concentrating with a 100 kD molecular weight cut-off ultrafiltration device (Pall, Shanghai, China), the sEVs were precipitated by ultracentrifugation at 110,000 × g at 4°C for 2 h using a Himac CP-90NX ultracentrifuge (Himac, Japan). After removing the supernatant, the pellet was resuspended and ultracentrifuged at 110,000 × g for another 2 h to further remove any remaining contaminants. Lastly, the purified sEV pellet was resuspended in 200 μl D-PBS and then stored at –80°C for further use. Briefly, total RNA was extracted from sEV samples for library construction. The RNA library was purified and separated from 18–30 nt RNA by PAGE electrophoresis, and then small RNA was reverse transcribed as cDNA and further amplified. After filtering by quality, the small RNA libraries were sequenced on the DNBSEQ platform. The raw data were evaluated and further filtered to obtain clean tags, which were stored in FASTQ format and used for next analysis.