GSM8195638: Naive-EVs, biol rep2; Homo sapiens; miRNA-Seq
Conditioned media from MSC cell cultures, treated with or without IFN-β treatment, after centrifuged them for 10 minutes at 2000 × g to remove any remaining cellular debris, the cleared supernatants were gathered and put into ultracentrifuge tubes (Polyallomer Quick-Seal centrifuge tubes 25 × 89 mm Beckman Coulter) and centrifuged for 30 minutes at 10000 × g (Type Ti 45, Beckman Coulter) in an ultracentrifuge (Optima L-90 K or Optima XE-90 Ultracentrifuge, Beckman Coulter) in order to extract macrovesicles. Subsequently, after meticulously gathering the supernatants, a Type 45 Ti rotor was used to centrifuge them for two hours at 110,000 × g and 4 °C. The EVs pellet obtained from this process was reconstituted in PBS and stored at −80 °C. Total RNA was extracted from exosome pellet using the TRIzol reagent (Invitrogen/Life Technologies) following the manufacturer's protocol. Individual libraries were constructed using 1µg of total RNA isolated from each sample. Each library was indexed in order to multiplex three samples per sequencing run on the Illumina Novaseq 6000 platform.