Sequences in the cytoplasmic tail of SARS-CoV-2 Spike facilitate expression at the cell surface and syncytia formation

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1. Version published to 10.1038/s41467-021-25589-1

   Sep 9, 2021
2. 

   Version published to 10.1101/2020.10.12.335562v2 on bioRxiv

   May 3, 2021
3. 

   ScreenIT

   Oct 15, 2020

   SciScore for 10.1101/2020.10.12.335562: (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	Contamination: Cells were free of mycoplasma as determined by regular testing (MycoAlert,

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Where indicated a primary anti-6xHis or anti-SNX27 antibody crosslinked to HRP was used.	anti-6xHis suggested: None
   The cells were washed twice with ice cold FACS buffer and incubated with an anti-AF488 antibody (1:67, A-11094	anti-AF488 suggested: None
   , Thermo Fischer Scientific; to quench non-internalised anti-HA AF488 conjugate), an anti-mouse AF647 antibody (1:300, Thermo Fischer …

   SciScore for 10.1101/2020.10.12.335562: (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	Contamination: Cells were free of mycoplasma as determined by regular testing (MycoAlert,

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Where indicated a primary anti-6xHis or anti-SNX27 antibody crosslinked to HRP was used.	anti-6xHis suggested: None
   The cells were washed twice with ice cold FACS buffer and incubated with an anti-AF488 antibody (1:67, A-11094	anti-AF488 suggested: None
   , Thermo Fischer Scientific; to quench non-internalised anti-HA AF488 conjugate), an anti-mouse AF647 antibody (1:300, Thermo Fischer Scientific, A31571; to relabel the non-internalised anti-HA conjugate) and an eFluor 780 fixable viability dye.	anti-mouse AF647 suggested: None
   Cells were incubated with an anti-calreticulin antibody (1:200, Abcam, ab2907) diluted in blocking buffer for 1 hour in order to counterstain the ER.	anti-calreticulin suggested: (Aviva Systems Biology Cat# ARP30113_T200, RRID:AB_387606)
   Cells were incubated for 1 hour in darkness with an anti-rabbit AF555 secondary antibody (1:300, Thermo Fischer Scientific, A31572) and an anti-HA AF488 conjugate (1:1000) diluted in blocking buffer with the latter intended to label HA-tagged S associated with intracellular membranes (as well as available external HA-S epitopes).	anti-rabbit suggested: (Molecular Probes Cat# A-31572, RRID:AB_162543)
   An anti-HA antibody (1:300, Roche, 3F10) diluted in ice cold media was added and cells were incubated on ice for 30 minutes.	3F10 suggested: None
   Cells were incubated with an anti-rat AF647 secondary antibody (1:300, Abcam, ab150154) diluted in 20% FCS in PBS to label non-internalised anti-HA antibody under non-permeabilising conditions at room temperature for 20 minutes.	anti-rat AF647 suggested: None anti-HA suggested: None
   Cells were incubated with an anti-SNX27 antibody (1:300, Abcam, ab77799) in blocking buffer for 1 hour in order to counterstain endosomes.	anti-SNX27 suggested: (Abcam Cat# ab77799, RRID:AB_10673818)
   Cells were incubated for 1 hour in darkness with an anti-mouse AF555 secondary antibody (1:300, Thermo Fischer Scientific, A31571) and an anti-rat AF488 secondary antibody (1:300, Thermo Fischer Scientific, A21208) diluted in blocking buffer with the latter intended to stain internalised anti-HA antibody.	anti-mouse suggested: (Molecular Probes Cat# A-31571, RRID:AB_162542) A31571 suggested: (Molecular Probes Cat# A-31571, RRID:AB_162542) anti-rat suggested: (Molecular Probes Cat# A-21208, RRID:AB_141709) A21208 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Comparison of internal and external levels of S by immunofluorescence: U2OS cells were seeded at a density of 2×104 cells/cm2 in 6-well plates in culture medium in a humidified incubator at 37°C with 5% CO2.	U2OS suggested: None
   Donor 293T cells were co-transfected with 1.5 µg of plasmids encoding different untagged S mutants and 0.6 µg of pmCherry-N1 using 6 µL of Fugene 6 following the manufacturer’s instructions (Promega)	293T suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
   Acceptor Vero cells were treated with CellTracker™ Green CMFDA (5-chloromethylfluorescein diacetate, Thermo Scientific) for 30 minutes.	Vero suggested: ATCC Cat# CCL-81.5, RRID:CVCL_1K11)
   Software and Algorithms
   Sentences	Resources
   Raw data files from LC-MS/MS data were were processed using Proteome Discoverer v2.1 (Thermo Scientific), and then searched against a human protein database (UniProt KB, reviewed) using the Mascot search engine programme	Proteome Discoverer suggested: (Proteome Discoverer, RRID:SCR_014477) Mascot suggested: (Mascot, RRID:SCR_014322)
   Analysis of mass spectral intensities: All raw files were processed with MaxQuant v1.5.5.1 using standard settings and searched against the UniProt Human Reviewed KB with the Andromeda search engine integrated into the MaxQuant software suite43,44.	MaxQuant suggested: (MaxQuant, RRID:SCR_014485)
   Enzyme search specificity was Trypsin/P for both endoproteinases.	Enzyme suggested: (ENZYME, RRID:SCR_002487)
   Missing values were imputed by values simulating noise using the Perseus default settings.	Perseus suggested: (Perseus, RRID:SCR_015753)
   Data was analysed using FlowJo v10 in which singlets were gated according to forward and side scatter profiles, dead cells were excluded using the viability dye and non-transfected cells were excluded based on their low AF488 and 647 signals.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   , Thermo Fischer Scientific; to quench non-internalised anti-HA AF488 conjugate), an anti-mouse AF647 antibody (1:300, Thermo Fischer Scientific, A31571; to relabel the non-internalised anti-HA conjugate) and an eFluor 780 fixable viability dye.	Thermo Fischer Scientific suggested: None

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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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4. 

   Version published to 10.1101/2020.10.12.335562v1 on bioRxiv

   Oct 12, 2020