Discovery and functional interrogation of SARS-CoV-2 protein-RNA interactions

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1. 

   ScreenIT

   Feb 25, 2022

   SciScore for 10.1101/2022.02.21.481223: (What is this?)

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

   Table 1: Rigor

   Ethics	IRB: This study protocol was approved by the Institutional Review Board of UCSD’s Human Research Protections Program (181180).
   Sex as a biological variable	not detected.
   Randomization	The foreground was a bed file of significant IDR peaks; the background was randomly defined peaks within the same annotated region as the foreground peaks.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Authentication: Cell culture and cell line generation: BEAS-2B, HEK293T and Vero E6 cells were purchased from the American Type Culture Collection and were not further authenticated. Contamination: Cells were routinely tested for mycoplasma contamination with a MycoAlert mycoplasma test kit …

   SciScore for 10.1101/2022.02.21.481223: (What is this?)

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

   Table 1: Rigor

   Ethics	IRB: This study protocol was approved by the Institutional Review Board of UCSD’s Human Research Protections Program (181180).
   Sex as a biological variable	not detected.
   Randomization	The foreground was a bed file of significant IDR peaks; the background was randomly defined peaks within the same annotated region as the foreground peaks.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Authentication: Cell culture and cell line generation: BEAS-2B, HEK293T and Vero E6 cells were purchased from the American Type Culture Collection and were not further authenticated. Contamination: Cells were routinely tested for mycoplasma contamination with a MycoAlert mycoplasma test kit (Lonza) and were found negative for mycoplasma.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The remaining lysates were immunoprecipitated using 15 µl anti-Strep or 10 µl anti-FLAG antibody (depending on the epitope tag of the construct; Supplementary Table 4) on Sheep Anti-Mouse IgG Dynabeads M-280 (ThermoFisher) overnight at 4°C.	anti-FLAG suggested: None Anti-Mouse IgG suggested: (Thermo Fisher Scientific Cat# 11201D, RRID:AB_2783640)
   Negative control samples are wild type (WT) BEAS-2B cells, and performed using both anti-Strep and anti-FLAG antibodies (separately).	anti-Strep suggested: None
   Cells were fixed with 4% paraformaldehyde for 30 min, which inactivates the virus, before transferring from BSL3 to BSL2, and proceeding with immunofluorescence staining using anti-Nucleocapsid antibody (40143-R019, Sino Biological).	BSL2 suggested: None anti-Nucleocapsid suggested: None
   Next, cells were incubated with primary antibodies (Supplementary Table 4) at 1:250-2000 dilutions in blocking buffer for 16 h at 4 °C, washed with PBS+0.01% Triton X-100 three times for 5 min each at room temperature, and then incubated with secondary antibody (goat anti-rabbit secondary IgG (H+L)	anti-rabbit suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell culture and cell line generation: BEAS-2B, HEK293T and Vero E6 cells were purchased from the American Type Culture Collection and were not further authenticated.	HEK293T suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
   The ACE2-overexpressing A549 cell line (A549-ACE2) was clonally generated and a gift from Benjamin tenOever52.	A549 suggested: None
   BEAS-2B cells were cultured on Matrigel (Corning) coated plates and maintained in the PneumaCult-Ex Plus Medium (Stem Cell Technologies), supplemented with 33 µg/ml hydrocortisone (Stem Cell Technologies).	BEAS-2B suggested: None
   HEK293T, Vero E6 and A549-ACE2 cells were cultured in DMEM (ThermoFisher) supplemented with 10% FBS (ThermoFisher) and passaged every three days.	Vero E6 suggested: None A549-ACE2 suggested: None
   SARS-CoV-2 virus infection: SARS-CoV-2 isolates USA-WA1/2020 (BEI Resources, #NR-52281), hCoV-19/USA/CA_UCSD_5574/2020 (lineage B.1.1.7) and hCoV-19/South Africa/KRISP-K005325/2020 (lineage B.1.351 BEI Resources NR-54009) were propagated and infectious units quantified by plaque assay using Vero E6 cells for the WA1 variant, and Vero-TMPRSS2 cells for variants B.1.1.7 and B.1.351.	Vero-TMPRSS2 suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Cell culture and cell line generation: BEAS-2B, HEK293T and Vero E6 cells were purchased from the American Type Culture Collection and were not further authenticated.	BEAS-2B suggested: None
   Recombinant DNA
   Sentences	Resources
   Plasmid construction: 2xStrep-tagged plasmids in a pLVX vector expressing SARS-CoV-2 proteins were a gift from Nevan Krogan1.	pLVX suggested: RRID:Addgene_174088)
   Cloning into the pcDNA3.4 was performed using FastDigest restriction enzymes EcoRI and BshT1 (Invitrogen) and Gibson assembly (NEB).	pcDNA3.4 suggested: RRID:Addgene_131198)
   SARS-CoV-2 ORFs were amplified by PCR (KAPA HiFi HotStart ReadyMix, Roche) from the 2xStrep-tagged or 3xFLAG-tagged plasmids with oligonucleotide primers containing attB recombination sites and recombined into pDONR221 using BP clonase II (ThermoFisher) (Supplementary Table 6).	pDONR221 suggested: RRID:Addgene_63743)
   Software and Algorithms
   Sentences	Resources
   This study protocol was approved by the Institutional Review Board of UCSD’s Human Research Protections Program (181180).	Human Research Protections Program suggested: None
   For reads mapped to the SARS-CoV-2 genome, bedgraph densities were generated using SAM tools v1.9 to obtain read densities at each nucleotide position.	SAM suggested: (SAM, RRID:SCR_010951)
   For reads mapped to the African Green Monkey genome, peaks were called on the usable reads by CLIPper54 and assigned to gene annotations in Ensembl ChlSab1.1 release 102 and Sars_cov_2 ASM985889 v3.101 were used to annotate peaks mapped to the African Green Monkey and SARS-CoV-2 genome.	Ensembl suggested: (Ensembl, RRID:SCR_002344)
   Gene Ontology analysis of eCLIP target genes was performed using ENRICHR (https://maayanlab.cloud/Enrichr/https://maayanlab.cloud/Enrichr/).	ENRICHR suggested: (Enrichr, RRID:SCR_001575)
   Cluster maps were visualized using Cytoscape version 3.8.1	Cytoscape suggested: (Cytoscape, RRID:SCR_003032)
   GENCODE v19 gene annotations and featureCounts (v.1.5.30) were used to create read count matrices.	featureCounts suggested: (featureCounts, RRID:SCR_012919)
   Sequencing reads are first processed as RNA-seq libraries, where RNA-seq reads were trimmed of adaptor sequences using cutadapt (v1.4.0) and mapped to repetitive elements (RepBase v18.04) using STAR (v2.4.0i).	STAR suggested: (STAR, RRID:SCR_004463)
   Multiple sequence alignment was performed using MAFFT v7.453 and default parameters, and sequence alignment was visualized using Jalview (version 1.0).	MAFFT suggested: (MAFFT, RRID:SCR_011811) Jalview suggested: (Jalview, RRID:SCR_006459)
   De novo motif analysis: HOMER was used to identify de novo motifs using reads from IDR peaks.	HOMER suggested: (HOMER, RRID:SCR_010881)
   The length of each region within the metagene was then scaled to 8%, 62% and 30%, corresponding to the average length of regions from the most highly expressed transcripts in ENCODE HepG2 RNA-seq control datasets.	ENCODE suggested: (Encode, RRID:SCR_015482)
   Interval features such as introns and exons were extracted from the Gencode v19 coordinates.	Gencode suggested: (GENCODE, RRID:SCR_014966)
   Images were collected via Zeiss ZEN software and converted to tiff for downstream analysis.	Zeiss ZEN suggested: None
   Images were analyzed using a custom-developed pipeline in CellProfiler (v.3.1.09).	CellProfiler suggested: None

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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: Please consider improving the rainbow (“jet”) colormap(s) used on page 64. 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:

   • 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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   Read the original source
2. 

   Version published to 10.1101/2022.02.21.481223v1 on bioRxiv

   Feb 24, 2022