| 生长方案: |
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. |
| 处理方案: |
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. |
| 提取方案: |
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. |
| 建库方案: |
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