SARS-CoV-2 expresses a microRNA-like small RNA able to selectively repress host genes

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1. Version published to 10.1073/pnas.2116668118

   Dec 13, 2021
2. 

   ScreenIT

   Sep 11, 2021

   SciScore for 10.1101/2021.09.08.459464: (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	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   After 30 min of cell lysis inside the BSC, the nuclei were pelleted and supernatants were transferred to 2.0 ml screw-top tubes with O-rings containing magnetic beads (SureBeads Protein G Magnetic Beads, NEB) coupled to antibodies (anti-pan Ago antibody, clone 2A8; Sigma Millipore). 10% of supernatants were kept for input (in TRIzol).	anti-pan Ago antibody , suggested: (Millipore Cat# MABE56, RRID:AB_10807962)
   Primary antibodies used were anti-FLAG M2 (Sigma Millipore)	anti-FLAG suggested: …

   SciScore for 10.1101/2021.09.08.459464: (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	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   After 30 min of cell lysis inside the BSC, the nuclei were pelleted and supernatants were transferred to 2.0 ml screw-top tubes with O-rings containing magnetic beads (SureBeads Protein G Magnetic Beads, NEB) coupled to antibodies (anti-pan Ago antibody, clone 2A8; Sigma Millipore). 10% of supernatants were kept for input (in TRIzol).	anti-pan Ago antibody , suggested: (Millipore Cat# MABE56, RRID:AB_10807962)
   Primary antibodies used were anti-FLAG M2 (Sigma Millipore)	anti-FLAG suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   PC-9 cells (kind gift from Dr. Craig Wilen) were cultured in RPMI medium (GIBCO) with 10% FBS and Pen/Strep. A549 (ATCC) cells were transduced as described previously (27) with hACE2 plasmid and cultured in F-12 medium (GIBCO) with 10% FBS, Pen/Strep and 1 μg/ml of puromycin (GIBCO).	PC-9 suggested: None A549 suggested: None
   Vero-E6 (ATCC), Huh7.5 and Huh7.5 Drosha knockout (kind gift from Dr. Charles Rice; (32)) cells were cultured in DMEM with 10% FBS, and Pen/Strep.	Huh7.5 suggested: RRID:CVCL_7927)
   Calu-3 cells were transduced either with FLAG-HA-Ago2 (27) or pLVX (for empty vector control) and cultured in the presence of 1 μg/ml of puromycin.	Calu-3 suggested: BCRJ Cat# 0264, RRID:CVCL_0609)
   Virus titers were determined by plaque assay using Vero-E6 cells.	Vero-E6 suggested: None
   Immunoprecipitation: For anti-pan Ago IP, 5×105 of Calu-3 cells or 1×105 of A549-hACE2 cells were infected with SARS-CoV-2 at MOI 5 for 24h.	A549-hACE2 suggested: RRID:CVCL_A5KB)
   Synthetic RNA transfections and qPCR: Synthetic RNAs (see Table 2 for sequences) were annealed by heating equimolar concentrations for 1 min at 90°C in siRNA buffer (Horizon, 60 mM KCl, 6 mM HEPES-pH 7.5, 0.2 mM MgCl2) and then incubating for 1 h at 37°C. 5×105 HEK293T cells were transfected with 30 μM of either vmiR-5p or control siRNA, by using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen).	HEK293T suggested: None
   Recombinant DNA
   Sentences	Resources
   PC-9 cells (kind gift from Dr. Craig Wilen) were cultured in RPMI medium (GIBCO) with 10% FBS and Pen/Strep. A549 (ATCC) cells were transduced as described previously (27) with hACE2 plasmid and cultured in F-12 medium (GIBCO) with 10% FBS, Pen/Strep and 1 μg/ml of puromycin (GIBCO).	hACE2 suggested: RRID:Addgene_1786)
   Calu-3 cells were transduced either with FLAG-HA-Ago2 (27) or pLVX (for empty vector control) and cultured in the presence of 1 μg/ml of puromycin.	pLVX suggested: RRID:Addgene_101121)
   The PCR products were cloned downstream of Renilla luciferase of psiCHECK(TM)-2 vector (Promega) using XhoI and NotI sites.	psiCHECK(TM)-2 suggested: None
   Cells were collected 6 h later, RNA extractions were performed using TRIzol and samples were processed for Northern blotting as described above. 2 4h later, 10 ng psiCHECK reporters and 2 μg pBlueScript II (Stratagene) were transfected using TransIT-293 Transfection Reagent (Mirus).	pBlueScript suggested: None
   Software and Algorithms
   Sentences	Resources
   After 3 days the supernatant was harvested and clarified by centrifugation, concentrated on Ultra-15 Centrifugal Filters (Amicon), aliquoted and stored at −80°C.	Amicon suggested: None
   The reads were mapped with bowtie2 (65) (--very-sensitive-local) to an index containing human and SARS-CoV-2 genomes.	bowtie2 suggested: (Bowtie 2, RRID:SCR_016368)
   miRNAs were counted by using featureCounts (66) and annotations obtained from miRbase (67).	featureCounts suggested: (featureCounts, RRID:SCR_012919) miRbase suggested: (miRBase, RRID:SCR_003152)
   Differential expression was determined using edgeR (68).	edgeR suggested: (edgeR, RRID:SCR_012802)
   Track visualization was performed using an IGV browser (69) of generated with BEDtools (70) bed files.	BEDtools suggested: (BEDTools, RRID:SCR_006646)
   Patient samples and quantitative RT-PCR: The human study reported here from which nasopharyngeal swab samples were obtained was approved by the Yale Human Research Protection Program, (Protocol 2000027971).	Yale Human Research Protection Program suggested: None
   Densitometry was performed by using Quantity One. miR-210-3p target and re-analysis of public data: Counts from RNA-seq experiments of lung biopsies were obtained from (14, 15).	Quantity One. suggested: None
   Primary antibodies used were anti-FLAG M2 (Sigma Millipore)	Sigma Millipore suggested: (Penn Cell Center Stockroom, RRID:SCR_010003)
   Target predictions: Custom Perl scripts that use the RNAduplex algorithm — part of the Vienna RNA Package (53) — were used to hybridize the vmiR-5p sequence to mRNA transcripts obtained from GENCODE (v38) (74) fragmented into 50-nt windows with 5-bp steps.	GENCODE suggested: (GENCODE, RRID:SCR_014966)

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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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   Results from scite Reference Check: We found no unreliable references.

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3. 

   Version published to 10.1101/2021.09.08.459464v1 on bioRxiv

   Sep 8, 2021