The SARS-CoV-2 RNA–protein interactome in infected human cells

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1. Version published to 10.1038/s41564-020-00846-z

   Dec 21, 2020
2. 

   ScreenIT

   Jul 16, 2020

   SciScore for 10.1101/2020.07.15.204404: (What is this?)

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

   Table 1: Rigor

   NIH rigor criteria are not applicable to paper type.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   For protein detection, we used the following primary antibodies: Nucleoprotein – Abcam #ab272852, POP1 – Proteintech #12029-1-AP.	POP1 suggested: (Proteintech Cat# 12029-1-AP, RRID:AB_2166477)
   We used the following secondary antibodies: IRDye 800CW Goat anti-Rabbit IgG (H + L) (LI-COR)	anti-Rabbit IgG suggested: None
   In the meantime, we coupled 100 μl Protein G Dynabeads (Thermo Fisher Scientific) with 10 μg CNBP antibody (Proteintech, 14717-1-AP) or 10 μg IgG antibody (Cell Signaling Technology, 2729) by incubating antibody-bead suspensions 45 minutes at room temperature.	CNBP suggested: (Proteintech Cat# …

   SciScore for 10.1101/2020.07.15.204404: (What is this?)

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

   Table 1: Rigor

   NIH rigor criteria are not applicable to paper type.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   For protein detection, we used the following primary antibodies: Nucleoprotein – Abcam #ab272852, POP1 – Proteintech #12029-1-AP.	POP1 suggested: (Proteintech Cat# 12029-1-AP, RRID:AB_2166477)
   We used the following secondary antibodies: IRDye 800CW Goat anti-Rabbit IgG (H + L) (LI-COR)	anti-Rabbit IgG suggested: None
   In the meantime, we coupled 100 μl Protein G Dynabeads (Thermo Fisher Scientific) with 10 μg CNBP antibody (Proteintech, 14717-1-AP) or 10 μg IgG antibody (Cell Signaling Technology, 2729) by incubating antibody-bead suspensions 45 minutes at room temperature.	CNBP suggested: (Proteintech Cat# 14717-1-AP, RRID:AB_2081548)
   Experimental Models: Cell Lines
   Sentences	Resources
   All subsequent manipulation steps were carried out as described in the eCLIP library preparation protocol80, starting with the reverse transcription of recovered RNA fragments. eCLIP: To facilitate eCLIP experiments in SARS-CoV-2 infected HuH-7 cells, we applied the previously described RAP-MS approach to purify all SARS-CoV-2-bound proteins prior to immunoprecipitating individual proteins from this pool.	HuH-7 suggested: None
   Quantification of ribosomal frameshifting: HEK293 cells were transiently transfected with either the control construct or the frameshifting construct of our dual-color EGFP-mCherry translation reporter outlined in Supplementary Figure 3a.	HEK293 suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
   Software and Algorithms
   Sentences	Resources
   Quantification and identification of peptides and proteins (RAP-MS and proteome): MS/MS spectra were searched on the Spectrum Mill MS Proteomics Workbench against a RefSeq-based sequence database containing 41,457 proteins mapped to the human reference genome (hg38) obtained via the UCSC Table Browser (https://genome.ucsc.edu/cgi-bin/hgTables) on June 29, 2018, with the addition of 13 proteins encoded in the human mitochondrial genome, 264 common laboratory contaminant proteins, 553 human non-canonical small open reading frames, 28 SARS-CoV-2 proteins obtained from RefSeq derived from the original Wuhan-Hu-1 China isolate NC_045512.290, and 23 novel unannotated SARS-CoV-2 ORFs whose translation is supported by ribosome profiling31, yielding a total of 42,337 proteins.	UCSC Table Browser suggested: None RefSeq suggested: (RefSeq, RRID:SCR_003496)
   We added NuPAGE LDS Sample Buffer (Thermo Fisher Scientifc) and incubated samples for 3 min incubation at 95°C.	Thermo Fisher Scientifc suggested: None
   Computational analyses: Gene set and pathway enrichment analysis: We performed a hypergeometric Gene Ontology (GO) enrichment analysis for the expanded SARS-CoV-2 interactome proteins using the Database for Annotation, Visualization and Integrated Discovery (DAVID) tool (https://david.ncifcrf.gov/tools.jsp) and applying default settings.	DAVID suggested: (DAVID, RRID:SCR_001881)
   Genes were ranked based on the product of the log2 fold change and the log10 moderated t-test P value between the SARS-CoV2 treatment and mock treatments. eCLIP and RNA sequencing analysis: Paired-end sequencing reads from (i) eCLIP experiments, or (ii) sequencing of crosslinked RNA fragments following RAP-MS, were trimmed using a custom python script that simultaneously identified the umi-molecular identifier (UMI) associated with each read.	python suggested: (IPython, RRID:SCR_001658)
   Next, we removed PCR duplicates using the UMI-aware deduplication functionality in Picard’s MarkDuplicates.	Picard’s suggested: None

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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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3. 

   Version published to 10.1101/2020.07.15.204404 on bioRxiv

   Jul 15, 2020