A novel cell culture system modeling the SARS-CoV-2 life cycle

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1. Version published to 10.1371/journal.ppat.1009439

   Mar 12, 2021
2. 

   ScreenIT

   Dec 14, 2020

   SciScore for 10.1101/2020.12.13.422469: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	Contamination: All cell lines were tested negative for mycoplasma.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   IFN-β, neutralizing antibody and drug treatment: To assess the antiviral efficacies of the materials, 1×104 Caco-2-N cells were seeded into 96-well plates.	IFN-β suggested: None
   The blots were exposed to primary antibodies anti-N (05-0154, AbMax), S (40589-T62, Sino Biological), β-Tubulin (CW0098, CWBIO), Flag (F7425, Sigma), ACE2 (10108-T24, Sino Biological) in 5% nonfat milk in 1×PBS containing 0.1% …

   SciScore for 10.1101/2020.12.13.422469: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	Contamination: All cell lines were tested negative for mycoplasma.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   IFN-β, neutralizing antibody and drug treatment: To assess the antiviral efficacies of the materials, 1×104 Caco-2-N cells were seeded into 96-well plates.	IFN-β suggested: None
   The blots were exposed to primary antibodies anti-N (05-0154, AbMax), S (40589-T62, Sino Biological), β-Tubulin (CW0098, CWBIO), Flag (F7425, Sigma), ACE2 (10108-T24, Sino Biological) in 5% nonfat milk in 1×PBS containing 0.1% Tween 20 for 2 h.	anti-N suggested: None β-Tubulin suggested: (CWBio Cat# CW0098, RRID:AB_2814800) CW0098 , CWBIO) , Flag ( F7425 suggested: None ACE2 suggested: (Enzo Life Sciences Cat# ALX-804-722-C100, RRID:AB_11180102)
   Cells were fixed with 4% paraformaldehyde in PBS, permeablized with 0.2% Triton X-100, and incubated with the rabbit polyclonal antibody against SARS-CoV nucleocapsid protein (Rockland, 200-401-A50, 1μg/ml) at 4 °C overnight.	SARS-CoV nucleocapsid protein suggested: (Creative Diagnostics Cat# DMAB8869, RRID:AB_2392503)
   After three washes, cells were incubated with the secondary goat anti-rabbit antibody conjugated with Alexa Fluor 555 (Thermo #A32732, 2 μg/ml) for 2 h at room temperature, followed by staining with 4’,6-diamidino-2-phenylindole (DAPI).	anti-rabbit suggested: (Thermo Fisher Scientific Cat# A32732, RRID:AB_2633281)
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell culture: HEK293T, Vero, Vero E6, A549 and Caco-2 cells were maintained in Dulbecco’s modified Eagle medium (DMEM) (Gibco, China) supplemented with 10% (vol/vol	HEK293T suggested: None Vero E6 suggested: None A549 suggested: None Caco-2 suggested: None
   Twenty micrograms of viral RNA and 20μg N mRNA were mixed and added to a 4-mm cuvette containing 0.4 mL of Caco-2-N cells (8×106) in Opti-MEM.	Caco-2-N suggested: None
   Antiviral screening: Twelve hours prior to infection for the antiviral screening 5 × 104 Caco-2-Nint cells were seeded in 96 well plates.	Caco-2-Nint suggested: None
   Software and Algorithms
   Sentences	Resources
   Heatmaps were drawn by using R package “pheatmap” (https://www.r-project.org).	https://www.r-project.org suggested: (R Project for Statistical Computing, RRID:SCR_001905)
   To quantify the junction-reads from subgenomic RNAs, the STAR2.7.5c was used for reads mapping.	STAR2.7.5c suggested: None
   The 50% inhibitory concentrations (IC50; compound concentration required to inhibit viral replication by 50% reduction of GFP positive cells) were determined using logarithmic interpolation using GraphPad Prism software version 7.0.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   Fixed cells were resuspended in PBS and analyzed by LSRFortessa SORP (BD Biosciences) and FlowJo software.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   RNA-seq dataset generated here can be found in the aforementioned NCBI Gene Expression Omnibus (	Gene Expression Omnibus suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)

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   Results from OddPub: Thank you for sharing your data.

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   Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   Results from rtransparent:

   • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
   • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
   • No protocol registration statement was detected.

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3. 

   Version published to 10.1101/2020.12.13.422469 on bioRxiv

   Dec 13, 2020