characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2016-09-06T17:01:23Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2016-03-03T11:20:10Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA3885203
characteristics.characteristic: External Id
characteristics.text: Laboratory of Molecular Medicine and Genomics
characteristics.characteristic: INSDC center name
characteristics.text: 2016-09-06T17:01:23Z
characteristics.characteristic: INSDC first public
characteristics.text: 2016-03-03T11:20:10Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS1072337
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-4502:Sample 13
characteristics.characteristic: Submitter Id
characteristics.text: 1_2
characteristics.characteristic: biological replicate
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: cell
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/GO_0005623
characteristics.characteristic: cell fraction
characteristics.text: non tumorigenic hepatocyte cell
characteristics.characteristic: cell type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: 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.
characteristics.characteristic: description
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_10090
characteristics.characteristic: organism
characteristics.text: control
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0001461
characteristics.characteristic: rna interference
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: Sample 13
characteristics.characteristic: title