| Construction Protocol | Non tumorigenic hepatocyte cells (3A cells [16]) were grown in RPMI 1640 medium supplemented with 10% FBS (GIBCO® Life Technology, Monza, Italy), 50 ng/ml EGF, 30 ng/ml IGF II (PeproTech Inc., Rocky Hill, NJ, USA), 10 μg/ml insulin (Roche, Mannheim, Germany) and Penicillin/Streptomycin, on collagen I (GIBCO® Life Technology, Monza, Italy) coated dishes. To obtain hepatocyte exosomes, cells were cultured for 48-72h hours in RPMI-1640 supplemented with 10% FBS depleted of bovine exosomes by overnight centrifugation at 100.000xg (Beckman Optima L80; Beckman Coulter, Italy). Hepatocyte exosomes were then prepared from supernatants by sequential centrifugation/ultracentrifugation. Cells conditioned media were centrifuged at 2000xg for 20 minutes and 10.000xg for 30 minutes, to remove cells debris. Cleared supernatants were passed through 0.22 m filter membranes, then ultracentrifuged at 100.000xg for 70 minutes, finally resuspended in PBS. Exosome size was determined by Nanoparticle Tracking Analysis [14] with Nanosight LM-10 (Malvern Instruments, Malverns, UK). Total RNA from cells and exosomes was isolated by Qiazol and miRNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany) following manufacturer’s protocols. RNA purity was assessed by spectrophotometric measure of OD260/OD280 ~2 and OD260/ OD230 >1.8 with Nanodrop 2000c Spectrophotometer (ThermoFisher) and RNA quantitation was performed with QubitTM and Qubit® RNA assay (Life Technologies, Monza, Italy). For miRNAseq experiments the quality of isolated RNA was determined with Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). RNA samples to be sequenced were purified from several independent cell cultures and exosome isolation and then pooled together. Indexed libraries were prepared from about 500 ng/ea. purified RNA pool with TruSeq Small RNA Sample Prep Kit (Illumina Inc., San Diego, CA), as described earlier [17]. Then, miRNA expression profiles were analyzed by smallRNA-Seq. Sized indexed miRNA libraries were gel purified and sequenced in triplicates on HiSeq2500 (Illumina Inc., San Diego, CA ) at a concentration of 10pM/lane for 50 plus 7 additional cycles for indices sequencing. |
FTP
