Sentinel cells enable genetic detection of SARS-CoV-2 Spike protein

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   ScreenIT

   Apr 24, 2021

   SciScore for 10.1101/2021.04.20.440678: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	IRB: All cells were maintained at 37°C with 5% CO2 Source of Primary Human T cells: Blood was obtained from Blood Centers of the Pacific (San Francisco, CA) as approved by the University Institutional Review Board.
   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
   Antibodies: Surface expressed proteins were assayed for using Alexa Fluor 647 Anti-Myc tag antibody (Cell Signaling Technologies #2233S)	Anti-Myc tag suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   HEK293T cells expressing ACE2 and TMPRSS2, a generous gift of Hannah S …

   SciScore for 10.1101/2021.04.20.440678: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	IRB: All cells were maintained at 37°C with 5% CO2 Source of Primary Human T cells: Blood was obtained from Blood Centers of the Pacific (San Francisco, CA) as approved by the University Institutional Review Board.
   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
   Antibodies: Surface expressed proteins were assayed for using Alexa Fluor 647 Anti-Myc tag antibody (Cell Signaling Technologies #2233S)	Anti-Myc tag suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   HEK293T cells expressing ACE2 and TMPRSS2, a generous gift of Hannah S Sperber and Dr. Satish Pillai, were cultured in DMEM High Glucose (Gibco #10569-010) supplemented with 10% FBS, 1% of Antibiotic-Antimycotic,	HEK293T suggested: None
   Generation of Stable Cell Lines: Lenti-X 293T cells (Takara Bio #632180) were seeded at approximately 7e5 cells/well in a 6-well plate to yield ~80% confluency the following day.	293T suggested: None
   Media was changed after 24 hours, and at 48 hours the viral supernatant was filtered through a 0.45μm PVDF filter and added to Jurkat or K562 cells seeded at approximately 1e5 cells/well in a 12-well plate.	Jurkat suggested: None K562 suggested: None
   Prior to the flow cytometry, cells were seeded at densities described below in a 96 well plates, using flat-bottom plates (Falcon #353072) for experiments involving BHK-21 cells and U bottom plates for all other experiments (Falcon #877217) and incubated for 24-72 hours as specified by the experiment.	BHK-21 suggested: None
   Recombinant DNA
   Sentences	Resources
   The following day cells were transfected with 1.5μg of transfer vector containing the desired expression cassette, and the lentiviral packaging plasmids pMD2	pMD2 suggested: None
   G (170ng) and pCMV-dR8.91 (1.33μg) using 10μl of Lipofectamine 2000 (Invitrogen #11668-027) according to manufacturer protocols.	pCMV-dR8.91 suggested: None
   Briefly, 293T cells were transfected with plasmid DNA (340 ng of Spike vector, 1μg CMV-Gag-Pol (pCMV-dΔR8.91), 125 ng pAdvantage (Promega), 1 μg Luciferase reporter (per 6-well plate)) for 48 h.	pCMV-dΔR8.91 suggested: None
   For positive control, Spike vector was replaced with pMD2.G and for negative control this vector was omitted.	pMD2.G suggested: RRID:Addgene_12259)
   Software and Algorithms
   Sentences	Resources
   For LCB1 and LCB3, protein sequences were translated to human optimized coding sequences using Benchling.	Benchling suggested: (Benchling, RRID:SCR_013955)
   All data were collected using FACSDiva (BD Biosciences) Flow Cytometry: Flow cytometry was performed using a LSR-Fortessa (BD Biosciences).	FACSDiva suggested: (BD FACSDiva Software, RRID:SCR_001456)
   The protein concentration was estimated based on the protein absorbance at 280nm with a spectrophotometer (Nanodrop One, Thermo), flash frozen, and stored in −80 °C.	Thermo suggested: (Thermo Xcalibur, RRID:SCR_014593)
   All data analysis was conducted using custom Python scripts, available on github (https://github.com/weinberz/sarsnotch).	Python suggested: (IPython, RRID:SCR_001658)
   Analysis was conducted in Jupyter59 and relied on numpy60, matplotlib, seaborn, pandas, SciPy61, scikit-learn62 and fcsparser.	matplotlib suggested: (MatPlotLib, RRID:SCR_008624)

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   Results from OddPub: Thank you for sharing your code and 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: 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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2. 

   Version published to 10.1101/2021.04.20.440678v1 on bioRxiv

   Apr 20, 2021