Illumina NovaSeq 6000 paired end sequencing; RNAseq analyses in HEK293T-ACE2 cells infected with SARS-CoV-2 (VOC Delta, 24hours) vs untreated cells
RNAseq analyses in HEK293T-ACE2 cells infected with SARS-CoV-2 (VOC Delta, 24hours) vs untreated cells
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).