Functional landscape of SARS-CoV-2 cellular restriction

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1. Version published to 10.1016/j.molcel.2021.04.008

   Jun 1, 2021
2. 

   ScreenIT

   Oct 4, 2020

   SciScore for 10.1101/2020.09.29.319566: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	Consent: HTBE cells were derived from one donor and all tissues used for isolation of these cells were obtained under informed consent and conform to HIPAA standards to protect the privacy of the donors’ personal health information.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	Contamination: All cells were tested and were confirmed to be free of mycoplasma contamination.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Antibodies: The antibodies used in this study include: Immunofluorescence: rabbit-anti-SARS-CoV-2 N antibody (gift from Kwok-Yung Yuen, University of Hong …

   SciScore for 10.1101/2020.09.29.319566: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	Consent: HTBE cells were derived from one donor and all tissues used for isolation of these cells were obtained under informed consent and conform to HIPAA standards to protect the privacy of the donors’ personal health information.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	Contamination: All cells were tested and were confirmed to be free of mycoplasma contamination.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Antibodies: The antibodies used in this study include: Immunofluorescence: rabbit-anti-SARS-CoV-2 N antibody (gift from Kwok-Yung Yuen, University of Hong Kong), mouse anti-HM1.24 (BST2) (a gift from Chugai Pharmaceutical Co., Kanagawa, Japan), rat anti-FLAG-AlexaFluor-488 (Biolegend, #637317), mouse anti-HA-AlexaFluor-594 (Biolegend, #901511), donkey anti-mouse-AlexaFluor-488 (Jackson ImmunoResearch, #715-545-150), donkey anti-mouse-Rhodamine-Red-X (Jackson ImmunoResearch, #715-295-150).	anti-HM1.24 suggested: None BST2 suggested: None anti-FLAG-AlexaFluor-488 suggested: None anti-HA-AlexaFluor-594 suggested: None anti-mouse-AlexaFluor-488 suggested: None anti-mouse-Rhodamine-Red-X suggested: None
   , rabbit monoclonal anti-β-actin antibody (Cell Signaling, #4970) and rabbit monoclonal anti-CoxIV antibody (Cell Signaling #4850).	anti-β-actin suggested: None anti-CoxIV suggested: None
   Anti-SARS-CoV-2 N rabbit serum was added for 1 h at room temperature, followed by three washes with 1xPBS and a 1-h incubation with Alexa Fluor 568-conjugated anti-rabbit secondary antibody (Thermo Fisher Scientific) diluted in 3% BSA.	Anti-SARS-CoV-2 N rabbit serum suggested: None Anti-SARS-CoV-2 N suggested: None anti-rabbit suggested: None
   Cells transfected with M, E, N and S-HA were stained overnight with mouse anti-HM1.24 (BST-2) antibody (diluted 1:300 in 1% BSA in PBS) at 4°C.	BST-2 suggested: (Novus Cat# NB 300-700, RRID:AB_530731)
   Experimental Models: Cell Lines
   Sentences	Resources
   Vero E6 (ATCC CRL-1586)	Vero E6 suggested: None
   HeLa (ATCC CRL-1586), and Huh7 (Apath LLC, Brooklyn) cells were maintained in cell growth media: Dulbecco’s modified eagle medium (DMEM, Gibco) supplemented with 10 % heat-inactivated fetal bovine serum (FBS, Gibco), 50 U/mL penicillin - 50 µg/mL streptomycin (Fisher Scientific), 1 mM sodium pyruvate (Gibco), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Gibco), and 1X MEM non-essential amino acids solution (Gibco).	Huh7 suggested: None
   HEK293T and HeLa cells stably expressing ACE2 (293T-ACE2/HeLa-ACE2) were generated by transducing HEK293T or HeLa cells with human ACE2-expressing lentiviruses, followed by selection of resistant cells with puromycin (InvivoGen) at 2 μg/ml for 14 days.	HEK293T suggested: None HeLa suggested: None
   RNA-seq experiments: HTBE and A549 cells were seeded overnight and then treated with 100 IU/ml universal interferon beta (IFN, R&D Systems), or left untreated.	A549 suggested: None
   Briefly, 293T cells at passage 10 were cultured in monolayer on matrigel-coated plates.	293T suggested: None
   Briefly, BHK-21/WI-2 cells (Kerafast, MA) transfected with SARS-CoV-2 S protein were inoculated with VSV-G pseudotyped ΔG-luciferase VSV (Kerafast, MA).	BHK-21/WI-2 suggested: RRID:CVCL_HB78)
   Each electroporation reaction consisted of 2.5×10^5 HeLa-ACE2 cells, 3.5 µL crRNPs, and 20 µL electroporation buffer.	HeLa-ACE2 suggested: None
   VLP assays: HEK-293T cells seeded in 6-well plates were transfected using Lipofectamine 2000 (Thermo-Fisher) with 625 ng each of plasmids encoding M-FLAG, E-V5, N-V5 (Fig 5D), or 500 ng of M, E, and N-V5 (Fig S2C), with or without 625 ng 3xFLAG-Orf7a or human codon-optimized HIV-1 Vpu (pVpHu from Klaus Strebel) with or without 75 ng BST2 (pcDNA3.1-BST-2 from Autumn Ruiz and Edward Stephens).	HEK-293T suggested: None
   Software and Algorithms
   Sentences	Resources
   The human hg38 reference genome and RefSeq gene annotation were used for spliced read alignment and gene assignment.	RefSeq suggested: (RefSeq, RRID:SCR_003496)
   The analysis software Columbus v2.5	Columbus suggested: None
   Towards this aim, we used a pipeline that employs a combination of scripts and Cytoscape applications.	Cytoscape suggested: (Cytoscape, RRID:SCR_003032)
   First, to explore the highest confidence interactions of “seed” proteins, we selected the STRING - Human Protein Links - High Confidence (Score >= 0.7) protein-protein interaction network available on NDEx as the “background” network (link provided below).	STRING suggested: (STRING, RRID:SCR_005223)
   Image brightness was adjusted using Adobe Photoshop CS3.	Adobe Photoshop suggested: (Adobe Photoshop, RRID:SCR_014199)

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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 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 10.1101/2020.09.29.319566v1 on bioRxiv

   Sep 30, 2020