SARS-CoV-2 Permissive glioblastoma cell line for high throughput antiviral screening

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1. Version published to 10.1016/j.antiviral.2022.105342

   Jul 1, 2022
2. 

   ScreenIT

   Feb 16, 2022

   SciScore for 10.1101/2022.02.13.480238: (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	Viral cytopathic effect and GFP expression were scored in a blinded manner at different time points post infection, and the supernatants were collected and stored at -20 °C. 2.10 Flow cytometry: For the cell surface staining of transfected HEK293T cells, cells were collected, washed in PBS, resuspended, transferred to tubes and samples were centrifuged in a cooled centrifuge (4 °C) at 500 g for 5’.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: , Human adenocarcinomic bronchial epithelial cells Calu3 (Cat. No. HTB-55) and human glioblastoma U87 MG cells (Cat. No. HTB-14) were obtained …

   SciScore for 10.1101/2022.02.13.480238: (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	Viral cytopathic effect and GFP expression were scored in a blinded manner at different time points post infection, and the supernatants were collected and stored at -20 °C. 2.10 Flow cytometry: For the cell surface staining of transfected HEK293T cells, cells were collected, washed in PBS, resuspended, transferred to tubes and samples were centrifuged in a cooled centrifuge (4 °C) at 500 g for 5’.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: , Human adenocarcinomic bronchial epithelial cells Calu3 (Cat. No. HTB-55) and human glioblastoma U87 MG cells (Cat. No. HTB-14) were obtained from ATCC as mycoplasma-free stocks and grown in Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific) supplemented with 10% (v / v) fetal bovine serum (FBS; HyClone)

   Table 2: Resources

   Antibodies
   Sentences	Resources
   goat anti-Rabbit IgG monoclonal antibody (Cat. n° 4414, Cell Signaling Technologies).	anti-Rabbit IgG suggested: (Cell Signaling Technology Cat# 4414, RRID:AB_10693544)
   After removal of the supernatant, cells were incubated with the primary (anti-Spike) antibody (30’ at 4 °C), washed in PBS, followed by a 30 min incubation at 4 °C with the secondary (labeled) antibody, and washed again.	anti-Spike suggested: None
   Samples were washed twice with Perm/Wash buffer before the addition of the primary (anti-Nucleocapsid) antibody (0.3 µg per sample).	anti-Nucleocapsid suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   CRL-1586), Human adenocarcinomic alveolar epithelial cells A549 (Cat. No. CCL-185)	A549 suggested: None
   SARS-CoV-2 viral stocks were prepared by inoculation of confluent Vero E6 cells as described in detail in a recent preprint report [not peer-reviewed] (16)	Vero E6 suggested: RRID:CVCL_XD71)
   2.4 Lentiviral transduction of ACE2: For lentivirus production, 50-70 % confluent HEK293FT cells in T-25 flasks were transfected with Lipofectamine LTX & PLUS Reagent (Thermo Fisher Scientific)	HEK293FT suggested: RRID:CVCL_6911)
   The next day, the transfected HEK293T cells were collected, resuspended, and counted on a Luna automated cell counter (Logos Biosystems), and administered to the U87.ACE2+ cells (20,000 cells/well).	HEK293T suggested: None
   One day prior to the experiment, U87.ACE2+ cells were seeded at 20,000 cells/well in 96 well-plates.	U87.ACE2+ suggested: None
   Recombinant DNA
   Sentences	Resources
   Monophosphoramidate prodrug Remdesivir GS-5734 (Cat. n° 469411000, Acros Organics); purified SARS-CoV-2 spike-specific monoclonal rabbit primary antibody R001 (Cat. n° 40592-R001, Sino Biological). 2.3 Plasmids: Second generation lentiviral support plasmids pMD2.	pMD2 suggested: None
   G and pSPAX2 were obtained from Addgene (www.addgene.org, Cat. n° 12259 and 12260, respectively).	pSPAX2 suggested: RRID:Addgene_12260)
   Using the NEBuilder HiFi DNA assembly cloning kit (NEB), ACE2, derived from a pcDNA3.1-hACE2 vector (Cat. n° 145033, Addgene), was inserted into a pLenti6.3 vector (Thermo Fisher Scientific) in which the Cytomegalovirus (CMV)	pcDNA3.1-hACE2 suggested: RRID:Addgene_145033) pLenti6.3 suggested: RRID:Addgene_120848)
   immediate-early enhancer/promoter region was replaced with a 0.6kb minimal Ubiquitous Chromatin Opening Element (UCOE) sequence and a Spleen Focus Forming Virus (SFFV) promoter derived from pMH0001 (Cat. n° 85969, Addgene).	pMH0001 suggested: RRID:Addgene_85969)
   An internal ribosomal entry site (IRES) was inserted behind the ACE2 sequence via restriction digestion of a pEF1a-IRES vector (Cat. n° 631970	pEF1a-IRES suggested: None
   ) containing ten betasheets of a modified mNeonGreen and a porcine teschovirus-1 2A peptide (P2A) coding sequence was inserted between the IRES sequence and the Blasticidin resistance cassette. pCAGGS.SARS-CoV-2_SΔ19_fpl_mNG2(11)_opt was generated through gibson assembly (NEBuilder, New England Biolabs) of a pCAGGS vector backbone cleaved using EcoRV-HF and HindIII-HF (New England Biolabs) and a PCR fragment of a codon-optimized SARS-CoV-2 Wuhan spike protein with a C-terminal 19 amino acid deletion as described in (18).	pCAGGS suggested: RRID:Addgene_127347)
   pcDNA3.1.mNG2(1-10) was generated through gibson assembly (NEBuilder, New England Biolabs) of a pcDNA3.1 vector (Thermo Fisher Scientific), amplified by PCR, and a PCR fragment of the first 10 betasheets of a modified mNeonGreen, synthesized by Genscript.	pcDNA3.1.mNG2 suggested: None pcDNA3.1 suggested: RRID:Addgene_79663)
   In parallel, HEK293T cells were transfected with transfection mixes containing 2.5 µg pCAGGS.SARS-CoV-2_SΔ19_fpl_mNG2(	pCAGGS.SARS-CoV-2_SΔ19_fpl_mNG2 suggested: None
   Software and Algorithms
   Sentences	Resources
   Signal intensities were quantified with Image Lab software v5.0 (Bio-Rad).	Image Lab suggested: (Image Lab Software, RRID:SCR_014210)
   Acquisition of all samples was done on a BD FACSCelesta flow cytometer (BD Biosciences) with BD FACSDiva v8.0.1 software.	BD FACSDiva suggested: (BD FACSDiva Software, RRID:SCR_001456)
   Flow cytometric data were analyzed in FlowJo v10.1 (Tree Star).	FlowJo suggested: (FlowJo, RRID:SCR_008520)

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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 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: 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 10.1101/2022.02.13.480238 on bioRxiv

   Feb 14, 2022