SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2

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1. Version published to 10.1016/j.cell.2020.09.033

   Nov 1, 2020
2. 

   ScreenIT

   Jul 15, 2020

   SciScore for 10.1101/2020.07.14.201616: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: All work with the SARS-CoV-2 was conducted in Biosafety Level-3 conditions either at the University of California San Diego or at the Eva J Pell Laboratory, The Pennsylvania State University, following the guidelines approved by the Institutional Biosafety Committees.
   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
   Antibodies used were, anti-spike antibody [1A9] (GeneTex, GTX632604), anti-Nucleocapsid antibody (GeneTex, GTX135357), Anti-HS (Clone F58-10E4) (Fisher Scientific, NC1183789), and Anti-ACE2 (Cell …

   SciScore for 10.1101/2020.07.14.201616: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: All work with the SARS-CoV-2 was conducted in Biosafety Level-3 conditions either at the University of California San Diego or at the Eva J Pell Laboratory, The Pennsylvania State University, following the guidelines approved by the Institutional Biosafety Committees.
   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
   Antibodies used were, anti-spike antibody [1A9] (GeneTex, GTX632604), anti-Nucleocapsid antibody (GeneTex, GTX135357), Anti-HS (Clone F58-10E4) (Fisher Scientific, NC1183789), and Anti-ACE2 (Cell signaling, 4355S).	anti-spike suggested: (GeneTex Cat# GTX632604, RRID:AB_2864418) Anti-HS suggested: (US Biological Cat# H1890, RRID:AB_10013601) NC1183789 suggested: (AMSBIO Cat# 370255-1, RRID:AB_10891554) Anti-ACE2 suggested: (Cell Signaling Technology Cat# 4355, RRID:AB_2797606)
   Secondary antibodies were, Anti-His-HRP (Genscript, A00612), Avidin-HRP (Biolegend, 405103), and Streptavadin-Cy5 (Thermo Fisher, SA1011).	Anti-His-HRP suggested: None Avidin-HRP suggested: (Boston Biochem Cat# A-115, RRID:AB_10695012) SA1011 suggested: None
   The membranes were blocked 1 hr at room temperature with Odyssey PBS Blocking Buffer (Li-Cor, 927-40000) or in 5% Blotting-Grade Blocker (Biorad, 1706404) in TBST (50 mM Tris buffer, pH 7.5 containing 150 mM NaCl and 0.1% Tween-20) and then incubated overnight at 4 °C with anti-hACE2 antibody (1:1000; R&D AF933) and anti-beta actin (1:2000; CST 4970) in 5% BSA (Sigma-Aldrich A9647) in TBST.	anti-hACE2 suggested: None anti-beta actin suggested: None
   The blot was incubated at room temperature for 1 hr with appropriate secondary antibodies (Donkey anti-Goat, Li-Cor, 926-32214; IRDye 690LT Donkey anti-Rabbit, Li-Cor, 926-68023; both at 1:20,000) in 5% BSA or 2.5% Blotting-Grade Blocker and 0.02% SDS in TBST.	anti-Goat suggested: (LI-COR Biosciences Cat# 926-32214, RRID:AB_621846) anti-Rabbit suggested: (LI-COR Biosciences Cat# 926-68023, RRID:AB_10706167)
   After 2 hr at 37°C, the inoculum was removed and cells were refed with DMEM supplemented with 10% FBS, 50 U/mL penicillin, 50 μg/mL streptomycin, and VSV-G antibody (I1, mouse hybridoma supernatant from CRL-2700; ATCC).	VSV-G suggested: None I1 suggested: None
   Cells were permeabilized for flow cytometry using BD Cytofix/Cytoperm according to the manufacturers protocol for fixed cells and stained with anti-spike antibody [1A9] (GeneTex GTX632604) and anti-Nucleocapsid antibody (GeneTex GTX135357) that were directly conjugated with Alexa Fluor 647 and Alexa Fluor 594 labeling kits (Invitrogen), respectively.	anti-Nucleocapsid suggested: (GeneTex Cat# GTX135357, RRID:AB_2868464)
   Experimental Models: Cell Lines
   Sentences	Resources
   Briefly, 100 mL of HEK293-6E cells were seeded at a cell density of 0.5 × 106 cells/ml 24 hr before transfection with polyethyleneimine (PEI).	HEK293-6E suggested: RRID:CVCL_HF20)
   Tissue Culture: NCI-H1299, A549, Hep3B, A375 and Vero E6 cells were from the American Type Culture Collection (ATCC).	NCI-H1299 suggested: None Hep3B suggested: None
   A375 wild-type and B4GALT7-/- cells were transfected with 15 μg ACE2 expression plasmid in a mixture of DMEM, OptiMEM (Gibco), Lipofectamine 2000 with Plus reagent (Invitrogen).	B4GALT7-/- suggested: None
   Media containing the lentivirus was collected and used to infect A549 WT and A375 WT cells, which were subsequently cultured with 5 μg/mL and 2 μg/mL blasticidin, respectively, to select for stably transduced cells.	A549 suggested: None A375 suggested: None
   The lentiviral sgRNA construct was generated in HEK293T cells, using the same protocol as for the Cas9 expression plasmid, and used to infect A549-Cas9 and A375-Cas9 cells to generate CRISPR knockout mutant cell lines.	HEK293T suggested: None
   Virus plaque assays: Confluent monolayers of Vero E6 or Hep3B cells were infected with SARS-CoV-2 at an MOI of 0.1.	Vero E6 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   A375 wild-type and B4GALT7-/- cells were transfected with 15 μg ACE2 expression plasmid in a mixture of DMEM, OptiMEM (Gibco), Lipofectamine 2000 with Plus reagent (Invitrogen).	A375 suggested: None
   Software and Algorithms
   Sentences	Resources
   Molecular Modeling: An electrostatic potential map of the SARS-CoV-2 spike protein RBD domain was generated from a crystal structure (PDB:6M17) and visualized using Pymol (version 2.0.6 by Schrödinger).	Pymol suggested: (PyMOL, RRID:SCR_000305)
   Particles on the micrographs were picked using DogPicker (Voss et al., 2009), stack with a box size of 200 pixels, and 2D classified with RELION 3.0 (Scheres, 2012).	RELION suggested: (RELION, RRID:SCR_016274)
   The cells were washed twice and then analyzed using a FACSCalibur or a FACSCanto instrument (BD Bioscience).	FACSCalibur suggested: None
   Data analysis was performed using FlowJo software and statistical analyses were done in Prism 8 (GraphPad).	FlowJo suggested: (FlowJo, RRID:SCR_008520) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
   Cells were permeabilized for flow cytometry using BD Cytofix/Cytoperm according to the manufacturers protocol for fixed cells and stained with anti-spike antibody [1A9] (GeneTex GTX632604) and anti-Nucleocapsid antibody (GeneTex GTX135357) that were directly conjugated with Alexa Fluor 647 and Alexa Fluor 594 labeling kits (Invitrogen), respectively.	BD Cytofix/Cytoperm suggested: None

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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: 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 10.1101/2020.07.14.201616v1 on bioRxiv

   Jul 14, 2020