Gene Expression Nebulas
A data portal of transcriptomic profiles analyzed by a unified pipeline across multiple species

Gene Expression Nebulas

A data portal of transcriptome profiles across multiple species

PRJNA631969: RNA-Seq of Human iPSC-cardiomyocytes infected with SARS-CoV-2

Source: NCBI / GSE150392
Submission Date: May 12 2020
Release Date:
Update Date: Oct 11 2021

Summary: Coronavirus disease 2019 (COVID-19) is a viral pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 is predominantly defined by respiratory symptoms, but cardiac complications including arrhythmias, heart failure, and viral myocarditis are also prevalent. Although the systemic ischemic and inflammatory responses caused by COVID-19 can detrimentally affect cardiac function, the direct impact of SARS-CoV-2 infection on human cardiomyocytes is not well understood.

Overall Design: We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a model system to examine the mechanisms of cardiomyocyte-specific infection by SARS-CoV-2. These studies show that SARS-CoV-2 can infect hiPSC-CMs in vitro, establishing a model for elucidating the mechanisms of infection and potentially a cardiac-specific antiviral drug screening platform.

GEN Datasets:
GEND000480
Strategy:
Species:
Healthy Condition:
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Cell Line:
Protocol
Growth Protocol: HiPSC-CM Differentiation: The iPSC differentiation work in the Svendsen Lab is carried out under ro00021505: Svendsen Stem Cell Program authorized by the Cedars-Sinai Medical Center IRB. The hiPSC-CMs were generated from hiPSCs using a small-molecule mediated differentiation approach that modulates Wnt signaling (Sharma et al., 2015). Briefly, this approach uses the CHIR99021 GSK3 inhibitor to initiate mesoderm specification, followed by Wnt-C59 Wnt inhibitor to initiate cardiac specification. Cells began beating at approximately day 7 post-differentiation. Cardiomyocytes were metabolically selected from other differentiated cells by using glucose deprivation as previously described (Sharma et al., 2015). After selection, hiPSC-CMs were replated as a monolayer into 96-well plate format at 100,000 cells per well in hiPSC-CM culture medium, RPMI 1640 + B27 supplement with insulin.
Treatment Protocol: SARS-CoV-2 infection of hiPSC-CMs: SARS-CoV-2, isolate USA-WA1/2020, was obtained from the Biodefense and Emerging Infections (BEI) Resources of the National Institute of Allergy and Infectious Diseases (NIAID). Importantly, all studies involving SARS-CoV-2 infection of hiPSC-CMs were conducted within a Biosafety Level 3 facility at UCLA. SARS-CoV-2 was passaged once in Vero-E6 cells and viral stocks were aliquoted and stored at -80oC. Virus titer was measured in Vero-E6 cells by TCID50 assay. Vero-E6 cells were cultured in DMEM growth media containing 10% fetal bovine serum, 2 mM L-glutamine, penicillin (100 units/ml), streptomycin (100 units/ml), and 10 mM HEPES. Cells were incubated at 37掳C with 5% CO2. For hiPSC-CM infection, viral inoculum (MOI of 0.01) was prepared using serum-free media. Culture media from each well containing hiPSC-CMs was removed and replaced with 250 碌L of prepared inoculum. For mock infection, serum-free media (250 碌L/well) alone was added. The inoculated plates were incubated for 1 hour at 37掳C with 5% CO2. The inoculum was spread by gently tilting the plate sideways at every 15 minutes. At the end of incubation, the inoculum was replaced with fresh hiPSC-CM culture medium. Cells remained at 37掳C with 5% CO2 for 72 hours before analysis.
Extract Protocol: The SMART-Seq V4 Ultra Low RNA Input Kit for Sequencing (Takara Bio USA, Inc., Mountain View, CA) was used for reverse transcription and generation of double stranded cDNA for library preparation using the Nextera XT Library Preparation kit (Illumina, San Diego, CA). Total RNA quality was analyzed via the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and RNA quantified using QubitTM(ThermoFisher Scientific, Waltham, MA) fluorometric quantitation.An input of 10 ng total RNA was used for oligo(dT)-primed reverse transcription, cDNA amplification, and cDNA cleanup. The cDNA was analyzed on the 4200 TapeStation (Agilent Technologies). Quantification of cDNA was performed using Qubit.cDNA normalized to 30 pg/ml was fragmented and sequencing primers added simultaneously.A limiting-cycle PCR added Index 1 (i7) adapters, Index 2 (i5) adapters, and sequences required for cluster formation on the sequencing flow cell. Indexed libraries were cleaned up, library size verification performed on the 4200 TapeStation, and library quantified via Qubit. Libraries were sequenced on a NextSeq 500 using with a 1x75 bp read length and coverage of ~60M reads/cell.
Library Construction Protocol: -
Sequencing
Molecule Type: rRNA- RNA
Library Source:
Library Layout: SINGLE
Library Strand: -
Platform: ILLUMINA
Instrument Model: Illumina NextSeq 500
Strand-Specific: Unspecific
Samples
Basic Information:
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Sequencing:
Assessing Quality:
Analysis:
Data Resource GEN Sample ID GEN Dataset ID Project ID BioProject ID Sample ID Sample Name BioSample ID Sample Accession Experiment Accession Release Date Submission Date Update Date Species Race Ethnicity Age Age Unit Gender Source Name Tissue Cell Type Cell Subtype Cell Line Disease Disease State Development Stage Mutation Phenotype Case Detail Control Detail Growth Protocol Treatment Protocol Extract Protocol Library Construction Protocol Molecule Type Library Layout Strand-Specific Library Strand Spike-In Strategy Platform Instrument Model Cell Number Reads Number Gbases AvgSpotLen1 AvgSpotLen2 Uniq Mapping Rate Multiple Mapping Rate Coverage Rate
Publications
Human iPSC-Derived Cardiomyocytes Are Susceptible to SARS-CoV-2 Infection.
Cell reports. Medicine . 2020-06-29 [PMID: 32835305]
Disease Modeling and Disease Gene Discovery in Cardiomyopathies: A Molecular Study of Induced Pluripotent Stem Cell Generated Cardiomyocytes.
International journal of molecular sciences . 2021-03-24 [PMID: 33805011]