characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2018-08-31T17:03:36Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-16T15:22:03Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA104700138
characteristics.characteristic: External Id
characteristics.text: Laboratory Molecular Medicine and Genomics University of Salerno
characteristics.characteristic: INSDC center name
characteristics.text: 2018-08-31T17:03:36Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-16T15:22:03Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS2301521
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-6599:Sample 4
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: MIA Paca-2
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0002236
characteristics.characteristic: cell line
characteristics.text: epithelial cell
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CL_0000066
characteristics.characteristic: cell type
characteristics.text: human
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: common name
characteristics.text: Protocols: The optimized gRNA construct, targeting ANXA1, and the Cas9 expression construct, pGS-gRNA- Cas9-Neo, were obtained from GenScript (USA). The ANXA1 exon located at the third coding exon was selected for guide RNA design. The gRNA recognizes the following target sequence 5′- GATCAGCGGTGAGCCCCTA -3′. For the establishment of ANXA1 KO cell lines, pGS-gRNA-Cas9-Neo containing neomycin resistance gene (Genscript, USA) was used. MIA PaCa-2 cells were transfected using Lipofectamine 2000 Reagent (Life technologies), according to the manufacturer′s instructions (www.lifetechnologies.com). One week later, the cells were subject to selection by 700 μg/ml neomycin (Euroclone). Cell clones were obtained following the limit dilution. Single cell colonies were selected, gradually expanded and analyzed by Western blotting to pick ANXA1 KO clones. The rescue of miR-196a expression was performed by mimic transfection as following reported. Briefly, PGS, ANXA1 KO MIA PaCa-2 cells were transfected with hsa-miR196a-5p UAGGUAGUUUCAUGUUGUUGGG mimic purchased from Dharmacon and used at a final concentration of 100 nM for 24 and 48 hours. The sequences were transfected using Lipofectamine 2000 Reagent (Life technologies), according to the manufacturer’s instructions. In order to test the transfection efficiency, for each experiment with mimic we performed a control co-transfecting the sequence of our interest with Tye 563 probe 1 nM (Integrated DNA Technologies; IA, USA). After 24 and 48 hours from the transfection, images of fluorescence signal have been taken at Integrated Live Cell Workstation Leica AF-6000 LX. MIA PaCa-2, human pancreatic cancer cells, were cultured in DMEM (Lonza) containing L-Glutamine 2 mM, 10% heat-inactivated fetal bovine serum (FBS; Lonza) and 2,5% heat inactivated horse serum (HS; Lonza). The medium was supplemented with antibiotics (10000 U/ml penicillin and 10 mg/ml streptomycin; Lonza). The cell line were purchased from ATCC (Rockville, USA) and were stained at 37°C in 5% CO2 -95% air humidified atmosphere. PGS MIA PaCa-2 cells Total RNA was extracted from PGS and ANXA1 KO MIA PaCa-2 cells using TRI Reagent® (Sigma-Aldrich). RNA concentration was assayed with a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific) and its quality assessed with the Agilent 4200 TapeStation with Agilent RNA ScreenTape Assay (Agilent Technologies). TruSeq smallRNA library Prep Kit (Illumina)
characteristics.characteristic: description
characteristics.text: carcinoma
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0000313
characteristics.characteristic: disease
characteristics.text: pGS-gRNA- Cas9-Neo; hsa-miR196a-5p
characteristics.characteristic: genotype
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: organism
characteristics.text: pancreas
characteristics.characteristic: organism part
characteristics.text: miR-196a overexpression
characteristics.characteristic: phenotype
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: Sample 4
characteristics.characteristic: title