Transcriptomic analysis of sorted lung cells revealed a proviral activity of the NF-κB pathway toward SARS-CoV-2

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1. Version published to 10.1016/j.isci.2023.108449

   Dec 1, 2023
2. 

   ScreenIT

   Mar 3, 2022

   SciScore for 10.1101/2022.02.25.481978: (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	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Cells were incubated with antibodies recognizing the spike protein of SARS-CoV-2 (anti-S2 H2 162, a kind gift from Dr. Hugo Mouquet, Institut Pasteur, Paris, France) and subsequently with secondary anti-human AlexaFluor-647 antibody (1:1000, A21455 Thermo) for 30 min at 4°C.	anti-S2 suggested: None anti-human AlexaFluor-647 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Viral stocks were produced by amplification on Vero E6 cells, for 72 h in DMEM 2% FBS.	Vero E6 suggested: …

   SciScore for 10.1101/2022.02.25.481978: (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	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Cells were incubated with antibodies recognizing the spike protein of SARS-CoV-2 (anti-S2 H2 162, a kind gift from Dr. Hugo Mouquet, Institut Pasteur, Paris, France) and subsequently with secondary anti-human AlexaFluor-647 antibody (1:1000, A21455 Thermo) for 30 min at 4°C.	anti-S2 suggested: None anti-human AlexaFluor-647 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Viral stocks were produced by amplification on Vero E6 cells, for 72 h in DMEM 2% FBS.	Vero E6 suggested: None
   SARS-CoV-2 infected and bystander cell-sorting and RNA extraction on fixed samples for RNA-seq: A549-ACE2 cells were seeded the day prior to infection.	A549-ACE2 suggested: None
   Software and Algorithms
   Sentences	Resources
   Data were acquired using Attune NxT Acoustic Focusing Cytometer (Thermo Fisher) and analyzed using FlowJo software.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Adaptors were trimmed with Trim Galore v0.6.4 [90] (wrapper for cutadapt v2.10 [91] and FastQC v0.11.9 [92]), with options --stringency 5 --trim-n -q 20 --length 20 --paired --retain_unpaired.	Trim Galore suggested: (Trim Galore, RRID:SCR_011847) FastQC suggested: (FastQC, RRID:SCR_014583)
   STAR v2.7.3a [93] was used to map the reads, with default parameters.	STAR suggested: (STAR, RRID:SCR_004463)
   Read coverage was computed for each strand with bamCoverage (deepTools v3.5.0 [95]) with options --binSize 1 --skipNAs --filterRNAstrand forward/reverse.	deepTools suggested: (Deeptools, RRID:SCR_016366)
   Scallop was run on each library, and the resulting annotations were merged using cuffmerge v1.0.0 [96], with gencode annotation (v32) as reference (-g option).	cuffmerge suggested: (Cuffmerge, RRID:SCR_015688)
   Then BEDtools v2.29.2 [97] was used to retain only intergenic and antisens transcripts regarding gencode annotation.	BEDtools suggested: (BEDTools, RRID:SCR_006646)
   Gene counts were normalized on ERCC counts, using estimateSizeFactorsForMatrix function from DESeq2.	DESeq2 suggested: (DESeq, RRID:SCR_000154)
   All plots were made using custom script, except for heatmaps that were done using pheatmap package (RRID:SCR_016418).	pheatmap detected: pheatmap ( RRID:SCR_016418)
   Gene expression quantification was performed using featureCounts v2.0.0 [98], with options -O -M --fraction -s 2, using a merged annotation of gencode v32, SARS-CoV-2 (NC045512.2) and newly annotated transcripts.	featureCounts suggested: (featureCounts, RRID:SCR_012919)
   GO enrichment analysis: The GO enrichment and KEGG pathway analysis were performed using DAVID online tool (updated version 2021) [101,102].	KEGG suggested: (KEGG, RRID:SCR_012773) DAVID suggested: (DAVID, RRID:SCR_001881)
   GOTERM_BP_DIRECT and KEGG pathway were retained and top 10 results based on adjusted p-value (Benjamini) were plotted using ggplot2 R package (v 3.3.0).	ggplot2 suggested: (ggplot2, RRID:SCR_014601)
   For identifying lncRNAs and unreferenced RNAs that possess NF-κB binding site in their promoter, we used p65 ChIP-seq data from GEO dataset GSE34329 [51] - one input file and 2 ChIP replicates, 38nt long reads, single-end.	ChIP-seq suggested: (ChIP-seq, RRID:SCR_001237)
   Reads were mapped using bowtie2 v2.4.1 using hg38 as reference, and SAMtools was used to retained the one flagged as primary alignment, with mapping quality > 30, and to remove PCR duplicates (markdup, with -r option).	bowtie2 suggested: (Bowtie 2, RRID:SCR_016368) SAMtools suggested: (SAMTOOLS, RRID:SCR_002105)
   NF-κB binding sites were then detected using macs2 v2.2.7.1 [103], with command callpeak -t ChIP_BamFile1 ChIP_BamFile2 -c input_BamFile -f BAM -g hs -s 38 --keep-dup all.	macs2 suggested: (MACS, RRID:SCR_013291)
   NF-kB motif genomic coordinates in the human genome were retrieved using EMBOSS fuzznuc v6.6 [104], using motif 5’-G(3)[AG]N[CT](3)C(2) - 3’ [105], on forward and reverse strand (option -complement Y).	EMBOSS suggested: (EMBOSS, RRID:SCR_008493)
   Statistical analysis was performed in GraphPad Prism 9 (GraphPad Software Inc.).	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

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

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3. 

   Version published to 10.1101/2022.02.25.481978 on bioRxiv

   Feb 26, 2022