Dynamics of SARS-CoV-2 host cell interactions inferred from transcriptome analyses

Article activity feed

1. 

   ScreenIT

   Jul 8, 2021

   SciScore for 10.1101/2021.07.04.450986: (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	Contamination: Cells have been tested negative for mycoplasma infection (MycoAlert Plus; Lonza, Basel, Switzerland). Authentication: Microscopy: To analyze SARS-CoV-2 N protein expression in Caco-2 cells by immunofluorescence, microscopic images were taken at 0, 4, 24 and 48 hpi using a Nikon Eclipse Ti-S fluorescence microscope (Nikon, Tokio, Japan).

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Reagents: We used antibodies to detect the SARS-CoV-2 nucleoprotein (mouse monoclonal; Sino Biologicals, Hong Kong, China) and GFP (mouse monoclonal; …

   SciScore for 10.1101/2021.07.04.450986: (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	Contamination: Cells have been tested negative for mycoplasma infection (MycoAlert Plus; Lonza, Basel, Switzerland). Authentication: Microscopy: To analyze SARS-CoV-2 N protein expression in Caco-2 cells by immunofluorescence, microscopic images were taken at 0, 4, 24 and 48 hpi using a Nikon Eclipse Ti-S fluorescence microscope (Nikon, Tokio, Japan).

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Reagents: We used antibodies to detect the SARS-CoV-2 nucleoprotein (mouse monoclonal; Sino Biologicals, Hong Kong, China) and GFP (mouse monoclonal; Roche Diagnostics).	GFP suggested: None
   For microscopy, the secondary antibody Alexa Fluor goat anti-mouse 568 (Thermo Fisher Scientific, Waltham, MA, USA) was applied.	anti-mouse 568 suggested: None
   A Horseradish peroxidase-conjugated anti-mouse antibody (Southern Biotech, Birmingham, AL, USA) was used for immunoblotting.	anti-mouse suggested: None
   Secondary antibody (anti-mouse CW800) and DNA dye Draq5 (Abcam, Cambridge, UK) were diluted 1/10,000 in blocking buffer and incubated for 1 h at RT.	anti-mouse CW800 suggested: None Draq5 suggested: (Biostatus Cat# DR50050, RRID:AB_2314341)
   Experimental Models: Cell Lines
   Sentences	Resources
   Caco-2, Vero E6 and HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal calf serum (Biochrom, Berlin, Germany), penicillin and streptomycin (100μg/ml; Invitrogen).	Caco-2 suggested: None Vero E6 suggested: None
   HEK293T cells were transfected with X-tremeGENE HP (Roche Diagnostics, Rotkreuz, Switzerland) following the manufacturer’s instructions.	HEK293T suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
   Recombinant DNA
   Sentences	Resources
   To generate a construct for co-expressing mCherry and SARS-CoV-2 main protease (3CLpro), a fragment encoding 3CLpro was first obtained as a double-stranded DNA fragment (gBlock; IDT, San José, CA, USA) and cloned into pcDNA3.1(−) via unique NheI/NotI restriction sites.	pcDNA3.1 suggested: RRID:Addgene_79663)
   The 3CLpro encoding vector was then linearized 5’ of the 3CLpro start codon via NheI and the mCherry-P2A fragment was inserted via Gibson Assembly, hence yielding a vector encoding mCherry-P2A-3CLpro.	mCherry-P2A-3CLpro suggested: None
   Software and Algorithms
   Sentences	Resources
   Paired-end sequencing of 2×150bp was performed at GeneWiz Inc. using an Illumina NovaSeq 6000 instrument (Illumina, San Diego CA, USA).	GeneWiz suggested: (GENEWIZ, RRID:SCR_003177)
   Live-cell experiments were performed on a Nikon Ti inverted microscope, equipped with a CSU-22 Yokogawa confocal spinning disc slider (Yokogawa Electric Corporation, Tokyo, Japan), a 60× Plan Apo NA 1.4 objective lens (Nikon), a Hamamatsu C9100-02 EMCCD camera (Hamamatsu Photonics, Hamamatsu, Japan), and the Volocity software (PerkinElmer; Waltham, MA, USA).	Volocity suggested: (Volocity 3D Image Analysis Software, RRID:SCR_002668)
   Microscopic images were evaluated with ImageJ software (NIH, Bethesda, MA, USA).	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   For read alignment, the STAR software was used with default settings (Dobin et al, 2013).	STAR suggested: (STAR, RRID:SCR_004463)
   Subsequently, BAM files were split by their reference using the SAMtools software suite (Li et al, 2009) and counted separately using the featureCounts function from the Subread package (Liao et al, 2019).	SAMtools suggested: (SAMTOOLS, RRID:SCR_002105) featureCounts suggested: (featureCounts, RRID:SCR_012919)
   Overrepresented GO terms were inferred using the ‘goana’ and ‘topGO’ function from the limma package (Ritchie et al, 2015).	limma suggested: (LIMMA, RRID:SCR_010943)
   The MATLAB toolbox PottersWheel was used for model fitting (Maiwald & Timmer, 2008).	MATLAB suggested: (MATLAB, RRID:SCR_001622)
   To this end, the following six model simplifications were tested: (1) description of A turnover by one turnover parameter instead of separate parameters for synthesis and degradation of A (Variant 4.0.1), (2) description of the synthesis of A by mass-action instead of Michaelis-Menten (MM) kinetics (Variant 4.0.2), (3) mass-action instead of MM-kinetics for describing synthesis of P (Variant 4.0.3), (4) mass-action kinetics for describing synthesis and degradation of A by single turnover parameter (Variant 4.0.4), (5) single parameter for describing turnover of A and mass-action instead of MM-kinetics for synthesis of P (Variant 4.0.5), (6) mass-action kinetics for synthesis of A as well as P.	Variant suggested: (VARIANT, RRID:SCR_005194)

   ---

   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).

   ---

   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).

   ---

   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.

   ---

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

   ---

   Read the original source
2. 

   Version published to 10.1101/2021.07.04.450986v1 on bioRxiv

   Jul 5, 2021