RNA-Protein Interaction Analysis of SARS-CoV-2 5′ and 3′ Untranslated Regions Reveals a Role of Lysosome-Associated Membrane Protein-2a during Viral Infection

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1. Version published to 10.1128/msystems.00643-21

   Aug 31, 2021
2. 

   ScreenIT

   Jan 7, 2021

   SciScore for 10.1101/2021.01.05.425516: (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	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Anti-Lamp2 (Cat. No. 49067), anti-Lamp1	Anti-Lamp2 suggested: (Cell Signaling Technology Cat# 49067, RRID:AB_2799349)
   (Cat no. 83506) and anti-P62 (Cat no. 8025s) antibodies were from Cell signalling Technology (Massachusetts, USA).	anti-P62 suggested: None
   Anti-GAPDH antibody (Cat No. SC-25778) was from Santacruz Biotechnology (Texas, USA).	Anti-GAPDH suggested: (Santa Cruz Biotechnology Cat# sc-25778, RRID:AB_10167668)
   Anti-Flag antibody (Cat no. A190-101) was …

   SciScore for 10.1101/2021.01.05.425516: (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	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Anti-Lamp2 (Cat. No. 49067), anti-Lamp1	Anti-Lamp2 suggested: (Cell Signaling Technology Cat# 49067, RRID:AB_2799349)
   (Cat no. 83506) and anti-P62 (Cat no. 8025s) antibodies were from Cell signalling Technology (Massachusetts, USA).	anti-P62 suggested: None
   Anti-GAPDH antibody (Cat No. SC-25778) was from Santacruz Biotechnology (Texas, USA).	Anti-GAPDH suggested: (Santa Cruz Biotechnology Cat# sc-25778, RRID:AB_10167668)
   Anti-Flag antibody (Cat no. A190-101) was from Bethyl laboratory (Texas, USA).	Anti-Flag suggested: None
   3-MA (Cat no. M9281) and Anti-HA antibody (Cat no. A190-101) was from Sigma (Missouri, USA)	Anti-HA suggested: None
   At each time point, whole cell extract was prepared in 2X laemmli buffer and equal amount of protein was resolved by 10% SDS-PAGE, followed by western blot using anti-Biotin antibody (Fig 1E).	anti-Biotin suggested: None
   Both input and pull down samples were resolved by SDS-PAGE, followed by western blot using anti-CUGBP1 and GAPDH antibodies.	anti-CUGBP1 suggested: None
   Goat anti-rabbit IgG HRP conjugated secondary antibody was used to detect the protein bands by chemiluminescence (Clarity ECL substrate, Cat no.170-5061, Biorad, California, USA).	anti-rabbit IgG suggested: None
   In brief, Huh7 and Vero E6 cells were fixed with 4% Paraformaldehyde for 10 min at room temp, followed by incubation in blocking buffer (4% BSA in PBS) for 1 hour at room temp and incubation with Lamp2 and LC3b antibodies (1:50 and 1:50 dilution, respectively) in antibody dilution buffer (3% BSA in PBS+0.1% Tween-20) for 16 hours, at 4°C.	LC3b suggested: None
   Coverslips were washed three times in PBS, followed by incubation with 1:500 dilution of goat anti-rabbit Alexa Fluor 488 and 1:500 dilution of goat anti-mouse Alexa Fluor 647 antibodies (Thermo Fisher Scientific, Massachusetts, USA) in antibody dilution buffer at room temp for 1 hour.	anti-rabbit suggested: None anti-mouse suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Mammalian cell culture and transfection: Vero E6 and HEK 293T cells were obtained from ATCC (Virginia, USA)	Vero E6 suggested: None
   For experiments involving siRNA mediated gene silencing, Huh7 or Vero E6 cells were seeded at 70-80% confluency on 12 well TC dishes and incubated overnight at 37°C, 5% CO2.	Huh7 suggested: None
   Production of 5’-300 and 3’-203 RNA from the pRMB constructs was verified by transfection of pRMB 5’-300 and pRMB 3’-203 plasmids along with pRMB vector into HEK293T cells followed by RT-PCR detection of the 5’-300 and 3’-203 RNA using above-mentioned primer pairs.	HEK293T suggested: None
   For SARS-CoV-2 infection studies, 200 µl of stock virus was diluted in serum free media to 2000 TCID50/ml and added to Vero E6/ Huh7 cell monolayer (seeded in 24 well plate) for 1 hour at 37°C, supplemented with 5% CO2.	Vero E6/ suggested: RRID:CVCL_YZ66)
   Recombinant DNA
   Sentences	Resources
   (Addgene plasmid # 107253; http://n2t.net/addgene:107253; RRID:Addgene_107253)],	detected: RRID:Addgene_107253)
   BASU RaPID plasmid (Addgene plasmid # 107250; http://n2t.net/addgene:107250; RRID:Addgene_107250)	detected: RRID:Addgene_107250)
   Addgene plasmid # 107252; http://n2t.net/addgene:107252; RRID:Addgene_107252) were gifted by Paul Khavari.	detected: RRID:Addgene_107252)
   pCDNA Lamp2b was a gift from Joshua Leonard (Addgene plasmid # 71292; http://n2t.net/addgene:71292; RRID:Addgene_71292) and pCDNA lamp2c was a gift from Janice Blum (Addgene plasmid # 89342; http://n2t.net/addgene:89342; RRID:Addgene_89342).	detected: RRID:Addgene_71292) detected: RRID:Addgene_89342)
   Software and Algorithms
   Sentences	Resources
   The virus-host RPPI dataset was visualized using Cytoscape (version 3.1.0) (24)	Cytoscape suggested: (Cytoscape, RRID:SCR_003032)

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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: Please consider improving the rainbow (“jet”) colormap(s) used on page 39. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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   Results from rtransparent:

   • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
   • 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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3. 

   Version published to 10.1101/2021.01.05.425516v1 on bioRxiv

   Jan 6, 2021