SARS-CoV-2 nucleocapsid protein adheres to replication organelles before viral assembly at the Golgi/ERGIC and lysosome-mediated egress

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1. Version published to 10.1126/sciadv.abl4895

   Jan 7, 2022
2. 

   ScreenIT

   Jun 17, 2021

   SciScore for 10.1101/2021.06.15.448497: (What is this?)

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

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	NA oil objective lens at 30-35 random positions for each sample.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   4.3 Antibodies: All the antibodies used in this study are reported in the tables below: 4.4 Cells and Viruses: Vero cells (ATCC CCL-81) were cultured under standard conditions (37°C and 5% CO2) in Dulbecco-modified MEM (Sigma Aldrich) supplemented with 10% heat-inactivated foetal bovine serum (Gibco), antibiotics/antimycotics (100 units/mL penicillin, 100 μg/mL streptomycin, 0.025 μg/mL Gibco Amphotericin B, Gibco) and 2 mM …

   SciScore for 10.1101/2021.06.15.448497: (What is this?)

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

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	NA oil objective lens at 30-35 random positions for each sample.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   4.3 Antibodies: All the antibodies used in this study are reported in the tables below: 4.4 Cells and Viruses: Vero cells (ATCC CCL-81) were cultured under standard conditions (37°C and 5% CO2) in Dulbecco-modified MEM (Sigma Aldrich) supplemented with 10% heat-inactivated foetal bovine serum (Gibco), antibiotics/antimycotics (100 units/mL penicillin, 100 μg/mL streptomycin, 0.025 μg/mL Gibco Amphotericin B, Gibco) and 2 mM L-glutamine (GlutaMAX, Gibco).	antibiotics/antimycotics suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   The day before transfection, Vero cells were seeded at 30% confluence in 8-well Ibidi μ-slides (catalogue n. 80826) in antibiotic-free medium.	Vero suggested: None
   Recombinant DNA
   Sentences	Resources
   4.5 Transfection of Vero cells: The four structural proteins of SARS-CoV-2 were expressed in Vero cells using a pEVAC vector backbone.	pEVAC suggested: None
   To generate RNA standards for qRT-PCR, a 97 nucleotide fragment of the spike ORF was cloned into the pJET1.2 vector (Invitrogen).	pJET1.2 suggested: RRID:Addgene_117131)
   Software and Algorithms
   Sentences	Resources
   The results of this experiment were reviewed and approved by the biosafety committee of the Department of Chemical Engineering of the University of Cambridge. 4.2 Chemicals: Methanol-free formaldehyde was purchased from Thermo Fisher Scientific; the ampoules were used immediately after opening and any leftover formaldehyde discarded.	Thermo Fisher Scientific suggested: (Thermo Fisher Scientific, RRID:SCR_008452)
   The microscope was controlled using the Zen software (version 2.6) and for acquisition of 16-bit images a pinhole size of 1.0 Airy unit (AU) for each channel, a scan speed of 5 (1.47 μs / pixel) and 4x averaging were used.	Zen suggested: None
   4.12 Deconvolution: Confocal images and deskewed light sheet microscopy data of expanded samples were deconvolved using the PSF Generator and DeconvolutionLab2 plugins in Fiji (47).	Fiji suggested: (Fiji, RRID:SCR_002285)
   Spearman’s rank coefficients were computed by ColocAnalyzer as: Here d = rank(I1(q)) – rank(I2(q)) is the difference between ranks computed for pixel q in channel 1 and in channel 2 independently.	ColocAnalyzer suggested: None
   4.17 Spot detection and analysis: Nucleocapsid protein, dsRNA and lysosome spot detection and analysis from microscopy images were performed by a customized MatLab routine.	MatLab suggested: (MATLAB, RRID:SCR_001622)
   The OpenCV and scikit-image Python libraries were used for the analysis.	OpenCV suggested: (OpenCV, RRID:SCR_015526) Python suggested: (IPython, RRID:SCR_001658)

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   Results from OddPub: Thank you for sharing your code.

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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 5. 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.
   • No funding statement was detected.
   • No protocol registration statement was detected.

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   Results from scite Reference Check: We found no unreliable references.

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   Read the original source
3. 

   Version published to 10.1101/2021.06.15.448497v1 on bioRxiv

   Jun 15, 2021