characteristics: ERC000011
characteristics: ENA-CHECKLIST
characteristics: 2022-12-01T00:20:18Z
characteristics: ENA-FIRST-PUBLIC
characteristics: 2022-12-01T00:20:18Z
characteristics: ENA-LAST-UPDATE
characteristics: SAMEA110411656
characteristics: External Id
characteristics: 1. Ceinge Biotecnologie Avanzate 2. Dipartimento di medicina molecolare e biotecnologie mediche - Universita di Napoli Federico II
characteristics: INSDC center name
characteristics: 2022-12-01T00:20:18Z
characteristics: INSDC first public
characteristics: 2022-12-01T00:20:18Z
characteristics: INSDC last update
characteristics: public
characteristics: INSDC status
characteristics: ERS12510012
characteristics: SRA accession
characteristics: E-MTAB-11973:HEK293-SARS1
characteristics: Submitter Id
characteristics: ArrayExpress
characteristics: broker name
characteristics: 293T
characteristics: cell line
characteristics: epithelial cell
characteristics: cell type
characteristics: human
characteristics: common name
characteristics: Protocols: For this study, we used HEK-293T cells (CRL-3216, ATCC) and HEK-293T stable clones overexpressing human angiotensin I converting enzyme 2 (ACE2) (HEK293T-ACE2). For the generation of HEK293T-ACE2 stable clones, HEK293T cells (at ~70% confluency) were transfected with 1 μg of DNA plasmid pCEP4-myc-ACE2 (#141185, Addgene) with X-tremeGENE 9 DNA Transfection Reagent (06365779001; Sigma-Aldrich) diluted with serum-free Dulbecco’s modified Eagle’s medium (41966-029; Gibco) to a concentration of 3 μl reagent/100 μl medium (3:1 ratio [μl]). The transfection complex was added to the cells (after 15 minutes of incubation) in a dropwise manner. Following forty-eight hours from transfection, the cell culture medium was changed, and the cells clones were selected using 800 mg/ml hygromycin. HEK-293T cells (CRL-3216, ATCC) and HEK-293T stable clones overexpressing human ACE2 (HEK293T-ACE2) were grown in a humidified 37°C incubator with 5% CO2. The cells were cultured in feeder-free conditions using Dulbecco’s modified Eagle’s medium (41966-029; Gibco) with 10% fetal bovine serum (10270-106; Gibco), 2 mM L-glutamine (25030-024; Gibco), and 1% penicillin/streptomycin (P0781; Sigma-Aldrich), with medium changed daily. HEK293T-ACE2 cells were infected with SARS-CoV-2 viral particles. Uninfected cells were used as the negative control of infection. After 24 hours of infection, the cells were lysed for RNAseq. These experiments were performed in a BLS3-authorized laboratory. Total RNA, isolated from HEK293T-ACE2 cells by using TRIzol RNA Isolation Reagent (#15596018; Ambion, Thermo Fisher Scientific) was quantified in a NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer (Thermo Scientific™), checked for purity and integrity and submitted to Macrogen Europe B.V. for sequencing. Libraries were prepared using the TruSeq Stranded mRNA Library Prep Kit according to the protocols recommended by the manufacturer (i.e., TruSeq Stranded mRNA Reference Guide # 1000000040498 v00).
characteristics: description
characteristics: embryo stage
characteristics: developmental stage
characteristics: overexpressing human ACE2
characteristics: genotype
characteristics: Severe acute respiratory syndrome coronavirus 2
characteristics: infect
characteristics: Homo sapiens
characteristics: organism
characteristics: Homo sapiens
characteristics: organism
characteristics: kidney
characteristics: organism part
characteristics: Homo sapiens
characteristics: scientific_name
characteristics: HEK293-SARS1
characteristics: title