title: GSM7113304: HN9.10 cells treated with 100 nmol/L ceramide, replicate 3 [2-2c_S2]; Mus musculus; miRNA-Seq
library_construction_protocol: Exosome Isolation: Exosomes released in the culture medium from untreated HN9.10e cells (control sample) and Cer-treated HN9.10e cells (experimental sample) were isolated after 48 hours of treatment. The supernatant was centrifuged at 2000g x 30 min, filtered with 0.22µm filter unit (MILLEX-GS from Millipore, Molsheim, France), in order to remove larger extracellular vesicles and apoptotic bodies, and concentrated with Pierce™ Protein Concentrators PES, 100K MWCO from Thermo Scientific™. Then, the retained in the filter was used to isolate exosomes with “total exosome isolation” kit from Invitrogen-Thermo Fisher Scientific (Waltham, MA, USA) by following the instructions of the company. Exosomal miRNA Isolation: The exosomes-containing pellet was resuspended in 200ul of PBS buffer, quantified using the Exosome Quantitation Kit (System Biosciences) and stored at −80 °C until use. Total RNA was extracted from 5×1010 exosomes using mirVana miRNA Isolation kit (Life Technologies) and miRNA concentration was determined by Qubit microRNA assay kit (Invitrogen). Sequencing libraries were prepared based on the QIAseq miRNA library kit (QIAGEN, Hilden, Germany), following the manufacturer's instructions. Final libraries were checked with both Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and Agilent Bioanalyzer DNA assay or Caliper (PerkinElmer, Waltham, MA). Quantified libraries were mixed at an equimolar ratio and sequenced on single-end 150 bp mode on NovaSeq 6000 (Illumina, San Diego, CA).