Low expression of EXOSC2 protects against clinical COVID-19 and impedes SARS-CoV-2 replication

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1. Version published to 10.26508/lsa.202201449

   Oct 14, 2022
2. 

   ScreenIT

   Mar 10, 2022

   SciScore for 10.1101/2022.03.06.483172: (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	Missing values were randomly imputed with a width of 0.3 and downshift of 1.8 from the standard deviation.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The membrane was blocked for 1 hour with constant rotation in 2.5% Milk-PBS 0.1% tween20 and incubated overnight at 4°C with the EXOSC2 antibody (Proteintech-66099-1-Ig) or GAPDH antibody (Cell Signaling-14C10) diluted in 2.5% Milk-PBS-0.1% Tween20.	EXOSC2 suggested: None GAPDH suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Calu-3 cell culture: Calu-3 cells were cultured in …

   SciScore for 10.1101/2022.03.06.483172: (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	Missing values were randomly imputed with a width of 0.3 and downshift of 1.8 from the standard deviation.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The membrane was blocked for 1 hour with constant rotation in 2.5% Milk-PBS 0.1% tween20 and incubated overnight at 4°C with the EXOSC2 antibody (Proteintech-66099-1-Ig) or GAPDH antibody (Cell Signaling-14C10) diluted in 2.5% Milk-PBS-0.1% Tween20.	EXOSC2 suggested: None GAPDH suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Calu-3 cell culture: Calu-3 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 (1:1) GlutaMAX™ (ThermoFisher Scientific) supplemented with 10% FBS, 1% NEAA, 1% sodium pyruvate, 1% penicillin-streptomycin and maintained at 37°C, 5% CO2 and passaged with TrypLE™ Express 1X (ThermoFisher Scientific) when ∼80% confluent.	Calu-3 suggested: None
   HEK293T cells were used for lentiviral production, plated at a density of 3 × 106 per 10 cm dish.	HEK293T suggested: None
   The titre of the SARS-CoV-2 stock was determined by plaque assay using Vero E6 cells.	Vero E6 suggested: RRID:CVCL_XD71)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   SARS-CoV-2 production: SARS-CoV-2 strain Victoria was produced by infecting Vero E6 cells at an MOI of 0.01 in DMEM supplemented with 2% FCS for 72 hours, until cytopathic effects were visible.	SARS-CoV-2 suggested: None
   Recombinant DNA
   Sentences	Resources
   Cloning and viral transduction: The human EXOSC2 open-reading frame was amplified from HEK293 cDNA using oligonucleotides: gctagcATGGCGATGGAGATGAGGC (fwd) and ctcgagTCCCTCCTGTTCCAAAAGCCT (rvs) and cloned as a NheI/XhoI PCR fragment into the NheI/XhoI restriction sites of a lentiviral self-inactivating transfer vector (SIN) containing a woodchuck hepatitis virus post regulatory element (W) to overexpress EXOSC2 under a PGK promoter (pLV_SIN-W-PGK-EXOSC2).	pLV_SIN-W-PGK-EXOSC2 suggested: None
   Cells were transfected using a calcium chloride transfection containing 0.5M calcium chloride (Sigma), 2X HEPES Buffered Saline (Sigma) and four lentiviral component plasmids; pCMV delta 8.2 (13 µg), pRSV-Rev (3 µg), pMD.G (3.75 µg) (Addgene) and pLV_SIN-W-PGK-EXOSC2 (13 µg) (Delgon et al., 2000)	pCMV suggested: RRID:Addgene_16459) pRSV-Rev suggested: RRID:Addgene_12253) pMD . G suggested: None
   TWe prepared a standard curve using the ORF9 nucleoprotein coding sequence cloned in pcDNA3. 10µg of plasmid was linearised with Apa1 at 37°C for 2 hours.	pcDNA3 suggested: RRID:Addgene_15475)
   EXOSC2 reconstitution: 5 µg of pLenti-EXOSC2, with 2.5 µg pR8.91 and 2.5 µg of VSV-G plasmids were transfected in a 15 cm dish of Hek293T cells at 80% confluency.	pLenti-EXOSC2 suggested: None pR8.91 suggested: None VSV-G suggested: RRID:Addgene_138479)
   Software and Algorithms
   Sentences	Resources
   The system was controlled by Xcalibur 3.0.63 (Thermo Fisher) and DCMSLink (Dionex).	Xcalibur suggested: (Thermo Xcalibur, RRID:SCR_014593)
   Protein group output files generated by MaxQuant were loaded into Perseus version 1.6.10.50.	MaxQuant suggested: (MaxQuant, RRID:SCR_014485) Perseus suggested: (Perseus, RRID:SCR_015753)
   The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD031611.	PRIDE suggested: (Pride-asap, RRID:SCR_012052)
   CRISPR-Cas9 editing: A sgRNA targeting exon 1 of EXOSC2 (5’-GAUACAAUCACUACGGACAC-3’) was designed using the CRISPOR tool (http://crispor.tefor.net/) (Concordet & Haeussler, 2018).	http://crispor.tefor.net/ suggested: (CRISPOR, RRID:SCR_015935)
   The intensity of EXOSC2 band against GAPDH loading control was assessed using ImageJ. qRT-PCR for EXOSC2: Calu-3 cells were lysed on ice using Tri Reagent (Sigma) for 5 minutes in RNase-free conditions.	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   Human transcriptome analysis: Raw Fastq files were trimmed for the presence of Illumina adapter sequences using Cutadapt v1.2.1 (Martin 2011).	Cutadapt suggested: (cutadapt, RRID:SCR_011841)
   Reads were aligned to hg19 transcripts (n=180,253) using Kallisto v0.46.0 (Bray et al. 2016) to produce gene-level TPM estimates by aggregating transcripts per gene.	Kallisto suggested: (kallisto, RRID:SCR_016582)
   Differential expression analysis was performed using edgeR (Robinson et al. 2010).	edgeR suggested: (edgeR, RRID:SCR_012802)

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   Results from OddPub: Thank you for sharing your 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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   Results from scite Reference Check: We found no unreliable references.

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3. 

   Version published to 10.1101/2022.03.06.483172v1 on bioRxiv

   Mar 7, 2022