A virus-specific monocyte inflammatory phenotype is induced by SARS-CoV2 at the immune-epithelial interface

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1. 

   ScreenIT

   Oct 2, 2021

   SciScore for 10.1101/2021.09.29.462202: (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	We first selected transcripts with relevant variation by comparing (i) the vector of inter-replicate coefficients of variation (CV), a reliable indicator of experimental noise (computed as the mean of all inter-replicate CVs, this for every gene) with (ii) the overall CV within the dataset (averaged from CV computed from 1,000 randomly picked dataset pairs, excluding replicates).
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: Cultures were verified to be free of mycoplasma contamination using MycoAlert Mycoplasma Detection Kit (Lonza).

   Table 2: Resources

   Antibodies
   Sentences	Resources
   After …

   SciScore for 10.1101/2021.09.29.462202: (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	We first selected transcripts with relevant variation by comparing (i) the vector of inter-replicate coefficients of variation (CV), a reliable indicator of experimental noise (computed as the mean of all inter-replicate CVs, this for every gene) with (ii) the overall CV within the dataset (averaged from CV computed from 1,000 randomly picked dataset pairs, excluding replicates).
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: Cultures were verified to be free of mycoplasma contamination using MycoAlert Mycoplasma Detection Kit (Lonza).

   Table 2: Resources

   Antibodies
   Sentences	Resources
   After blocking the non-specific binding sites by incubation in blocking buffer (PBS + 10% of donkey serum) for 1 h, cells were stained (2 h, RT) with either: anti-GFP AlexaFluor488 (clone FM264G, BioLegend, #338008, 1:200)	anti-GFP suggested: (BioLegend Cat# 338008, RRID:AB_2563288)
   For EBOV and CoV2, cells were stained 30 min with the secondary antibodies anti-rabbit and anti-goat AlexaFluor488, respectively (both 1:500, Jackson ImmunoResearch).	anti-rabbit suggested: None anti-goat suggested: (Jackson ImmunoResearch Labs Cat# 005-500-003, RRID:AB_2337011)
   The cells were incubated overnight at 4°C with a rabbit antibody recognizing CoV2 N protein (Rockland, #200-401-A50; 1:1000) and a goat-anti-EBOV VP35 antibody (custom-made by Antagene, 1:200) in Cov2 and EBOV infection respectively.	#200-401-A50 suggested: (Rockland Cat# 200-401-A50, RRID:AB_828403) VP35 suggested: (Thermo Fisher Scientific Cat# PA5-112029, RRID:AB_2866765)
   The cells were washed 4 times in PBS and incubated with goat anti-rabbit antibody conjugated with AlexaFluor488 or donkey-anti-goat antibody conjugated with AlexaFluor594 for 1 h at RT (Invitrogen; 1:200). 4’,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) was used at 200 ng/mL for nuclei staining.	donkey-anti-goat suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Viruses: SARS-CoV2 stocks (isolate USA_WA1/2020), Influenza A PR8-GFP virus (A/Puerto Rico/8/1934(H1N1) and EBOV (isolate Mayinga) were grown in Vero E6 cells and purified by sucrose ultracentrifugation and titers determined.	Vero E6 suggested: RRID:CVCL_XD71)
   For Transwell, HEK cells were transfected and re-plated onto Transwell inserts.	HEK suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
   , hereafter IAV) and Madin-Darby canine kidney (MDCK) cells were provided by D. Lingwood (Ragon Institute).	MDCK suggested: CLS Cat# 602280/p823_MDCK_(NBL-2, RRID:CVCL_0422)
   Epithelial cells, in suspension (Caco-2) or seeded 24 h prior (HEK), at a concentration of 25.103 cells/well, were transfected with 100 ng of each plasmid using Lipofectamine™ 3000 reagent (Thermo Fisher) in Opti-MEM™ medium (Gibco) and washed 8 – 12 h later.	Caco-2 suggested: None
   Recombinant DNA
   Sentences	Resources
   Most proteins were cloned in a pLVX-Puro backbone under an EF1a promoter; Spike was cloned in a pTwist backbone under an EF1a promoter.	pLVX-Puro suggested: RRID:Addgene_154877) pTwist suggested: RRID:Addgene_164438)
   Software and Algorithms
   Sentences	Resources
   Flow cytometry was performed on a FACSymphonyTM flow cytometer (BD Biosciences) and analysed using FlowJo 10 software. Immunohistochemistry: For CoV2 and EBOV, Caco-2 cells seeded in 96-well plates were infected as described above.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   RNA-seq data processing and QC: Reads were aligned to the human genome (GENCODE GRCh38 primary assembly and gene annotations v27) with STAR 2.5.4a (https://github.com/alexdobin/STAR/releases).	GENCODE suggested: (GENCODE, RRID:SCR_014966)
   The gene-level quantification was calculated by featureCounts (http://subread.sourceforge.net/).	featureCounts suggested: (featureCounts, RRID:SCR_012919) http://subread.sourceforge.net/ suggested: (Subread, RRID:SCR_009803)
   Raw read counts tables were normalized by median of ratios method with DESeq2 package from Bioconductor (https://bioconductor.org/packages/release/bioc/html/DESeq2.html) (5) and then converted to GCT and CLS formats.	DESeq2 suggested: (DESeq, RRID:SCR_000154) Bioconductor suggested: (Bioconductor, RRID:SCR_006442) https://bioconductor.org/packages/release/bioc/html/DESeq2.html suggested: (DESeq2, RRID:SCR_015687)
   Reads were aligned by STAR 2.5.4a, with, for SARS-CoV2, specific parameters suggested in Kim et al.(6).	STAR suggested: (STAR, RRID:SCR_004463)
   Finally, the RNA integrity for all samples was measured by median Transcript Integrity Number (TIN) across human housekeeping genes with RSeQC software (http://rseqc.sourceforge.net/#tin-py).	RSeQC suggested: (RSeQC, RRID:SCR_005275)
   dn signature in Gene Ontology (GO) biological processes pathways and Reactome pathways was computed using Fisher’s exact test with BH-FDR correction, through the g:Profiler interface.	Reactome suggested: (Reactome, RRID:SCR_003485) g:Profiler suggested: (G:Profiler, RRID:SCR_006809)
   In order to overcome the inherent redundancy of GO pathways, significant pathways were then interpreted and visualized as an enrichment network using Cytoscape (v3.8.2) (7) and its EnrichmentMap and AutoAnnotate modules.	EnrichmentMap suggested: (EnrichmentMap, RRID:SCR_016052)
   Briefly, Cytoscape allows collapsing of redundant significant pathways into a single biological theme using the Jaccard Overlap combined index (cutoff=0.5), thus simplifying the interpretation.	Cytoscape suggested: (Cytoscape, RRID:SCR_003032)
   Regarding the type I IFN and IFN gamma signatures, data were downloaded from the Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/) from two human published datasets GSE142672 and GSE46599 (8).	Gene Expression Omnibus suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)
   Analyses and plots were done using S-Plus (v8.2.0), RStudio (v.1.2.5019) and GraphPad Prism (v.8.4.3)	RStudio suggested: (RStudio, RRID:SCR_000432) GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

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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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 56 and 57. 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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   Results from scite Reference Check: We found no unreliable references.

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2. 

   Version published to 10.1101/2021.09.29.462202 on bioRxiv

   Sep 29, 2021