SARS-CoV-2 nucleocapsid protein binds host mRNAs and attenuates stress granules to impair host stress response

Version published to 10.1016/j.isci.2021.103562

Jan 1, 2022

SciScore for 10.1101/2020.10.23.342113: (What is this?)

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

Table 1: Rigor

Institutional Review Board Statement	not detected.
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
The supernatant was transferred to a new tube and incubated with 1μg of GFP antibody for 4 hours to overnight, and subsequently 10 μl protein G Dyna beads were added and incubated for an additional 2 hours (Invitrogen catalogue number 10003D).	GFP suggested: None
Primary antibodies were used at 1: 5000 dilution, and horseradish peroxidase-conjugated goat anti-mouse (Thermo Fisher 31430) or antirabbit secondary (Thermo Fisher 31460) antibodies were used at …

SciScore for 10.1101/2020.10.23.342113: (What is this?)

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

Table 1: Rigor

Institutional Review Board Statement	not detected.
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
The supernatant was transferred to a new tube and incubated with 1μg of GFP antibody for 4 hours to overnight, and subsequently 10 μl protein G Dyna beads were added and incubated for an additional 2 hours (Invitrogen catalogue number 10003D).	GFP suggested: None
Primary antibodies were used at 1: 5000 dilution, and horseradish peroxidase-conjugated goat anti-mouse (Thermo Fisher 31430) or antirabbit secondary (Thermo Fisher 31460) antibodies were used at 1:10,000.	anti-mouse suggested: (Thermo Fisher Scientific Cat# 31430, RRID:AB_228307) antirabbit suggested: None
GFP-tagged viral proteins were immunoprecipitated with anti-GFP antibody (G10362, Life Technologies) overnight followed by a 2-hour incubation with Protein G Dynabeads (Invitrogen).	G10362 suggested: (Thermo Fisher Scientific Cat# G10362, RRID:AB_2536526)
Santa Cruz G3BP (H-10) antibody was used for staining at 1:100 concentration in block solution for 2 hours at room temperature (RT).	H-10 suggested: (Syd Labs Cat# PA000293-010516H10-0.1mg, RRID:AB_2074929)
Experimental Models: Cell Lines
Sentences	Resources
Cell cultures: HEK293 cells (Flp-In 293 T-REx cell lines) were obtained from Life Technologies (Invitrogen catalogue number R780-07).	293 suggested: None
The vectors were co-transfected into Flp-In T-REx 293 cells together with the pOG44 Flp recombinase expression plasmid.	Flp-In T-REx 293 suggested: RRID:CVCL_U427)
The AP-MS data generated from HEK293 cells expressing GFP alone were used as negative control for SAINTexpress analysis.	HEK293 suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
Software and Algorithms
Sentences	Resources
Human protein reference sequences from the UniProt Swiss-Prot database was downloaded on 18-06-2020.	UniProt suggested: (UniProtKB, RRID:SCR_004426)
The resulting data were filtered using SAINTexpress26 to obtain confidence values utilizing our two biological replicates.	SAINTexpress26 suggested: None
MS data visualization, functional enrichment and archiving: We used Cytoscape (V3.4.0;47) to generate protein-protein interaction networks.	Cytoscape suggested: (Cytoscape, RRID:SCR_003032)
Imaging was performed the next day using a Zeiss confocal spinning disc AxioObserverZ1 microscope equipped with an Axiocam 506 camera using Zen software.	Zen suggested: None
Lowest RIN was 9.3; median RIN score was 9.75. 1000 ng per sample was then processed using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (cat # E7760L; New England Biolabs, Ipswich, USA; protocol v. v3.1_5/20) including PolyA selection, with 15 minutes of fragmentation at 94 °C and 8 cycles of amplification.	PolyA suggested: (PolyA DB, RRID:SCR_007867)
Quantification and Statistical Analysis: RNA-seq Analysis: To identify the differentially expressed genes from RNA-seq data, we used DESeq258 (ver 3.11) on gene counts generated using STAR and the human Gencode annotation V19.	STAR suggested: (STAR, RRID:SCR_015899)
To plot differentially expressed genes as volcano plots, we used R-package EnhancedVolcanoplot (https://github.com/kevinblighe/EnhancedVolcano).	R-package suggested: None EnhancedVolcanoplot suggested: None
Additionally, GO/ KEGG enrichment was performed using ShinyGO (v0.61), which utilizes hypergeometric distribution followed by FDR correction where FDR cutoff was set to 0.05. iCLIP Metagene and Motif Analysis: Significant iCLIP peaks were called using Pureclip60, with input control and default settings, and the cross-linking sites within 50 bps from each other were merged.	KEGG suggested: (KEGG, RRID:SCR_012773)

Results from OddPub: Thank you for sharing your data.

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

Results from JetFighter: We did not find any issues relating to colormaps.

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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SARS-CoV-2 nucleocapsid protein binds host mRNAs and attenuates stress granules to impair host stress response

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