SARS-CoV-2 sensing by RIG-I and MDA5 links epithelial infection to macrophage inflammation

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1. 

   ScreenIT

   Jan 2, 2021

   SciScore for 10.1101/2020.12.23.424169: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: The study was approved by the joint University College London/University College London Hospitals NHS Trust Human Research Ethics Committee and written informed consent was obtained from all participants. Consent: The study was approved by the joint University College London/University College London Hospitals NHS Trust Human Research Ethics Committee and written informed consent was obtained from all participants.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Cells were then incubated with 1μg/ml CR3009 …

   SciScore for 10.1101/2020.12.23.424169: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: The study was approved by the joint University College London/University College London Hospitals NHS Trust Human Research Ethics Committee and written informed consent was obtained from all participants. Consent: The study was approved by the joint University College London/University College London Hospitals NHS Trust Human Research Ethics Committee and written informed consent was obtained from all participants.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Cells were then incubated with 1μg/ml CR3009 SARS-CoV-2 cross-reactive antibody (a kind gift from Dr. Laura McCoy,) in permeabilisation buffer for 30 min at room temperature, washed once and incubated with secondary Alexa Fluor 488-Donkey-anti-Human IgG (Jackson Labs).	488-Donkey-anti-Human IgG suggested: None
   A blocking step was carried out for 1h at room temperature with 10% goat serum/1%BSA in PBS. Nucleocapsid (N) proten detection was performed by primary incubation with human anti-N antibody (Cr3009, 1ug/ml) for 18h, and washed thoroughly in PBS.	anti-N suggested: None Cr3009 suggested: None
   Primary antibodies were detected by labelling with with secondary anti-human AlexaFluor488 and anti-rabbit AlexaFluor546 conjugates (Jackson Immuno Research) for 1h.	anti-rabbit suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Vero.E6 were provided by NIBSC, Beas2B (ATCC CRL-9609) and Hulec5a (ATCC CRL-3244) were obtained from ATCC, and Detroit 562 (ATCC CCL-138) were a kind gift from Dr Caroline Weight (UCL).	Hulec5a suggested: ATCC Cat# CRL-3244, RRID:CVCL_0A11)
   THP-1 cells were cultured in RPMI (Gibco) supplemented with 10 heat-inactivated FBS (Labtech)	THP-1 suggested: None
   Caco-2 and Calu-3 cells were stimulated for 24 h with media containing TLR4 agonist Lipopolysaccharide (LPS) (Peprotech), the TLR3 agonist poly I:C (Peprotech) or the TLR7 agonist R837 (Invivogen), using the concentration stated on each figure.	Calu-3 suggested: None
   Virus culture and infection: SARS-CoV-2 strain BetaCoV/Australia/VIC01/2020 (NIBSC) was propagated by infecting Caco-2 cells at MOI 0.01 TCID50/cell, in DMEM supplemented with 2% FBS at 37°C.	Caco-2 suggested: None
   Virus titres were determined by 50% tissue culture infectious dose (TCID50) on Vero.E6 cells.	Vero.E6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Software and Algorithms
   Sentences	Resources
   All samples were acquired on a BD Fortessa X20 or LSR II using BD FACSDiva software.	BD FACSDiva suggested: (BD FACSDiva Software, RRID:SCR_001456)
   Data was analysed using FlowJo v10 (Tree Star).	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Image analysis: NF-κB, IRF3, IL6 and GAPDH raw image channels were pre-processed using a batch rolling ball background correction in FIJI imagej software package (Schindelin et al., 2012) prior to quantification.	imagej suggested: (ImageJ, RRID:SCR_003070)
   Statistical analysis: Statistical analysis was performed using GraphPad Prism.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

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   Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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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 did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 29. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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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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2. 

   Version 1 published on bioRxiv

   Dec 23, 2020