organism: Homo sapiens
description: 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)
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2018-08-31T17:03:36Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-16T15:22:03Z
attributes.tag: External Id
attributes.value: SAMEA104700139
attributes.tag: INSDC center name
attributes.value: Laboratory Molecular Medicine and Genomics University of Salerno
attributes.tag: INSDC first public
attributes.value: 2018-08-31T17:03:36Z
attributes.tag: INSDC last update
attributes.value: 2018-03-16T15:22:03Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6599:Sample 5
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: MIA Paca-2
attributes.tag: cell type
attributes.value: epithelial cell
attributes.tag: common name
attributes.value: human
attributes.tag: disease
attributes.value: carcinoma
attributes.tag: genotype
attributes.value: pGS-gRNA- Cas9-Neo; hsa-miR196a-5p
attributes.tag: organism part
attributes.value: pancreas
attributes.tag: phenotype
attributes.value: miR-196a overexpression
attributes.tag: sample name
attributes.value: E-MTAB-6599:Sample 5
attributes.tag: scientific_name
attributes.value: Homo sapiens