Global analysis of protein-RNA interactions in SARS-CoV-2-infected cells reveals key regulators of infection

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1. Version published to 10.1016/j.molcel.2021.05.023

   Jul 1, 2021
2. 

   ScreenIT

   Mar 1, 2021

   SciScore for 10.1101/2020.11.25.398008: (What is this?)

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

   Table 1: Rigor

   NIH rigor criteria are not applicable to paper type.

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   Experimental model and subject detail: Virus growth kinetic experiments: 1.2 x 105 Calu-3 cells were seeded into each well of a 24-well plate.	Calu-3 suggested: None
   Briefly, 2.5 x 105 Vero cells were seeded into each well of a 24-well plate and cells were inoculated with 10-fold serial dilutions of SARS-CoV-2 containing supernatants for 1 h at 37°C.	Vero suggested: None
   Software and Algorithms
   Sentences	Resources
   To determine the infection rate, the number of infected cells at each time point was determined using the dsRNA fluorescence signal with Fiji software using a custom macro …

   SciScore for 10.1101/2020.11.25.398008: (What is this?)

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

   Table 1: Rigor

   NIH rigor criteria are not applicable to paper type.

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   Experimental model and subject detail: Virus growth kinetic experiments: 1.2 x 105 Calu-3 cells were seeded into each well of a 24-well plate.	Calu-3 suggested: None
   Briefly, 2.5 x 105 Vero cells were seeded into each well of a 24-well plate and cells were inoculated with 10-fold serial dilutions of SARS-CoV-2 containing supernatants for 1 h at 37°C.	Vero suggested: None
   Software and Algorithms
   Sentences	Resources
   To determine the infection rate, the number of infected cells at each time point was determined using the dsRNA fluorescence signal with Fiji software using a custom macro (Schindelin et al., 2012)	Fiji suggested: (Fiji, RRID:SCR_002285)
   3D-stacked images were taken with voxel size of 80 nm x 80 nm x 200 nm in x:y:z and images were deconvolved with maximum likelihood algorithm using cellSens (5 iterations, default PSF, no noise reduction, Olympus).	cellSens suggested: None
   Fluorescence intensity profiles were obtained using ImageJ “Plot profile” tool across 8 µm regions on 0.4 µm max intensity z-projected images.	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   Peptides were loaded onto a trap-column (Thermo Scientific PepMap 100 C18, 5 μm particle size, 100A pore size, 300 μm i.d. x 5mm length) and separation of peptides was performed by C18 reverse-phase chromatography at a flow rate of 300 nL/min and a reverse-phase nano Easy-Spray column (Thermo Scientific PepMap C18	PepMap suggested: (BioWorks, RRID:SCR_014594)
   Protein identification and quantification were performed using Andromeda search engine implemented in MaxQuant (1.6.3.4) (Cox 2011) under default parameters (Cox et al., 2011).	MaxQuant suggested: (MaxQuant, RRID:SCR_014485)
   Statistical analysis for the processed intensities was performed in R-package “limma (3.38.3)” using empirical Bayesian method moderated t-test.	R-package suggested: None “limma suggested: None
   Gene Ontology (GO) terms: Using the GO annotation available via the GO.db R package (3.11.4)	GO.db suggested: None
   GO enrichment analysis was performed using PANTHER classification system (http://www.pantherdb.org).	PANTHER suggested: (PANTHER, RRID:SCR_004869)
   Kyoto Encyclopedia of Genes and Genomes (KEGG)	KEGG suggested: (KEGG, RRID:SCR_012773)
   For each gene symbol in our dataset the number of PubMed articles matching with a search query “(SYMBOL) AND (virus)” where SYMBOL is the gene name, such as EIF4E were retrieved.	PubMed suggested: (PubMed, RRID:SCR_004846)

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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 52. 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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3. 

   Version published to 10.1101/2020.11.25.398008v2 on bioRxiv

   Nov 29, 2020
4. 

   ScreenIT

   Nov 26, 2020

   SciScore for 10.1101/2020.11.25.398008: (What is this?)

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

   Table 1: Rigor

   NIH rigor criteria are not applicable to paper type.

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   Lentiviruses were produced by cotransfection of HEK293T cells with pHEF-VSVG (NIH AIDS Research & Reference reagent program #4693) and psPAX2 (kind gift N. Proudfoot, Oxford, UK).	HEK293T suggested: None
   Briefly, 2.5 x Vero cells were seeded into each well of a 24-well plate and cells were inoculated with 10-fold serial dilutions of SARS-CoV-2 containing supernatants for 1 h at 37°C.	Vero suggest…

   SciScore for 10.1101/2020.11.25.398008: (What is this?)

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

   Table 1: Rigor

   NIH rigor criteria are not applicable to paper type.

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   Lentiviruses were produced by cotransfection of HEK293T cells with pHEF-VSVG (NIH AIDS Research & Reference reagent program #4693) and psPAX2 (kind gift N. Proudfoot, Oxford, UK).	HEK293T suggested: None
   Briefly, 2.5 x Vero cells were seeded into each well of a 24-well plate and cells were inoculated with 10-fold serial dilutions of SARS-CoV-2 containing supernatants for 1 h at 37°C.	Vero suggested: None
   Cell viability assay and determination of infection rate To establish cell viability and infection rate, 1.2 x 105 Calu-3 cells were seeded into each well of a 24-well plate onto glass coverslips.	Calu-3 suggested: None
   2x105 Calu3 cells were seeded on the dried coverslips and incubated in growth media for 48 hours prior to the experiment.	Calu3 suggested: None
   Software and Algorithms
   Sentences	Resources
   Nuclei were counted with a custom-made macro for the Fiji software package (Schindelin et al., 2012).	Fiji suggested: (Fiji, RRID:SCR_002285)
   3D-stacked images were taken with voxel size of 80 nm x 80 nm x 200 nm in x:y:z and images were deconvolved with maximum likelihood algorithm using cellSens (5 iterations, default PSF, no noise reduction, Olympus).	cellSens suggested: None
   Background subtraction was performed on all channels using rolling ball subtraction method (radius = 250 px) in ImageJ (National Institutes of Health).	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   Peptides were loaded onto a trap-column (Thermo Scientific PepMap 100 C18, 5 μm particle size, 100A pore size, 300 μm i.d. x 5mm length) and separation of peptides was performed by C18 reverse-phase chromatography at a flow rate of 300 nL/min and a reverse-phase nano Easy-Spray column (Thermo Scientific PepMap C18, 2μm particle size, 100A pore size, 75μm i.d. x 50cm).	Thermo Scientific PepMap suggested: (BioWorks, RRID:SCR_014594)
   For cRIC and WCP samples, MaxQuant search was performed with “match between run” activated.	MaxQuant suggested: (MaxQuant, RRID:SCR_014485)
   Statistical analysis for the processed intensities was performed in R-package “limma (3.38.3)” using empirical Bayesian method moderated ttest.	R-package suggested: None “limma suggested: None
   Gene Ontology (GO) terms Using the GO annotation available via the GO.db R package (3.11.4), GO terms including the term 'RNA binding' (to annotate RNA-binding related functions, processes, or compartments) or term 'immun' or exact terms 'immune response' and 'innate immune response' (to annotate immunity related functions, processes, or compartments) were selected.	GO.db suggested: None
   GO enrichment analysis was performed using PANTHER classification system (http://www.pantherdb.org).	PANTHER suggested: (PANTHER, RRID:SCR_004869)
   Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways KEGG pathways under the 'Immune system' category in the high-level KEGG hierarchy available via the R package “KEGGREST” (1.28.0) were selected (see tableS9) and genes mapping to these pathways were identified using “org.Hs.eg.db.” Pfam RNA-binding domains Classification of proteins into classical and non-classical RNA-binding proteins is based on their Pfam domain composition.	KEGG suggested: (KEGG, RRID:SCR_012773)
   PubMed literature linking genes to viral infections To automatically query the NCBI Entrez Utilities REST API, the R package “rentrez” (1.2.2) was used.	PubMed suggested: (PubMed, RRID:SCR_004846)
   Table 1 Package/Software DEP 1.4.1 DESeq2 1.28.1 DGIdb Oct2020 ggrepel 0.8.2 GO.db 3.11.4 htseq-count 0.11.3 KEGGREST	DESeq2 suggested: (DESeq, RRID:SCR_000154)
   limma 3.38.3 org.	limma suggested: (LIMMA, RRID:SCR_010943)
   b SRA toolkit STAR aligner 2.7.3a Stats 4.0.2 tidyverse suite 1.3.0 viridis 0.5.1 VSN 3.50.0 Source/Identifier DOI: 10.18129/B9.bioc.	STAR suggested: (STAR, RRID:SCR_015899)

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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.

   ---

   Results from TrialIdentifier: No clinical trial numbers were referenced.

   ---

   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: Please consider improving the rainbow (“jet”) colormap used on page 52. 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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   About SciScore

   SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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5. 

   Version published to 10.1101/2020.11.25.398008v1 on bioRxiv

   Nov 25, 2020