GSM5129981: Sample-05: f hAFS-EVs_normoxia-Donor3; Homo sapiens; miRNA-Seq
hAFS-EVs were isolated by serial ultracentrifugation from concentrated hAFS-CM. Briefly, hAFS-CM was collected and centrifuged at 4°C at 300×g for 10', 2000×g for 20' to remove cell debris. Surnatant was then processed at 10,000×g for 40'. Pellet was then discarded and surnatant further processed by ultracentrifugation in an Optima L-90K (Beckmann Coulter, Brea, California) at 100,000×g for 120' using Beckman Coulter's swinging-bucket SW55Ti centrifuge rotors. The pellet containing heterogenous hAFS-EVs was washed in PBS by a final step at 100,000×g for 120' and then re-suspended in 0,22 µm filtered PBS. hAFS-CM and hAFS-EV protein concentration were measured using the BiCinchoninic Acid (BCA) assay (Thermo Fisher Scientiﬁc, Wal-tham, Massachusetts). EVs isolated from hAFs were suspended in 700 µL of Qiazol reagent (Qiagen,). EV RNA was extracted with the miRNeasy Micro Kit (Qiagen) according to the manufacturer's instructions. miRNA content was assessed by performing the Qubit microRNA Assay Kit and RNA quality and sizes were checked with Agilent 2100 Bioanalyzer using the Agilent Small RNA chip according to manufacturer's protocol. Each sequencing library was constructed from 9.2ng of isolated miRNA. Small RNA libraries were prepared and amplified using QIAseq miRNA Library kit (Qiagen), following manufacturer's instructions. Libraries were pooled after quality check and quantification by TapeStation (Agilent Technologies) using Agilent High Sensitivity D1000 ScreenTape. Pooled libraries were quantified by real-time qPCR following “Sequencing Library qPCR Quantification” Guide (Illumina Inc.) and sequenced by Illumina NextSeq platform using the 75 cycles high output kit (Illumina Inc.).