Human organoid systems reveal in vitro correlates of fitness for SARS-CoV-2 B.1.1.7

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   May 8, 2021

   SciScore for 10.1101/2021.05.03.441080: (What is this?)

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

   Table 1: Rigor

   Ethics	IRB: The Medical Ethical Committee of the Erasmus MC Rotterdam granted permission for this study (METC 2012-512). Consent: Tissue from the ileum was obtained from the UMC Utrecht with informed consent of the patient, who was operated for resection of an intestinal tumor. IACUC: The study was approved by the UMC Utrecht (Utrecht, The Netherlands) ethical committee and was in accordance with the Declaration of Helsinki and according to Dutch law.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: Cell lines were tested negative for mycoplasma.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Next, …

   SciScore for 10.1101/2021.05.03.441080: (What is this?)

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

   Table 1: Rigor

   Ethics	IRB: The Medical Ethical Committee of the Erasmus MC Rotterdam granted permission for this study (METC 2012-512). Consent: Tissue from the ileum was obtained from the UMC Utrecht with informed consent of the patient, who was operated for resection of an intestinal tumor. IACUC: The study was approved by the UMC Utrecht (Utrecht, The Netherlands) ethical committee and was in accordance with the Declaration of Helsinki and according to Dutch law.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: Cell lines were tested negative for mycoplasma.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Next, cells were incubated in FACS buffer (2mM EDTA, 2.5% bovine serum albumin (BSA) in PBS) on ice for 5 min, stained with the AT2 marker antibody HTII-280 (1:40; Terrace Biotech) on ice for 15 min, and with goat anti-mouse IgM Alexa Fluor 488 (1:400; Invitrogen) for 5 min.	AT2 suggested: None anti-mouse IgM suggested: None
   Cells were incubated with primary antibodies overnight at 4°C in blocking buffer, washed twice with PBS, incubated with corresponding secondary antibodies (Alexa 488-/ 594-conjugated anti-rabbit IgG, anti-mouse IgG, or anti-mouse IgG1 (1:500; Invitrogen)) in blocking buffer for 2 hours at room temperature, washed two times with PBS, incubated with indicated additional stains (hoechst (ThermoFisher) or phalloidin (Santa Cruz)), washed twice with PBS, and mounted in Prolong Antifade (Invitrogen) mounting medium.	anti-mouse IgG suggested: None Antifade ( Invitrogen ) mounting medium . suggested: None
   For ACE2 stainings, cells were blocked with 1% BSA and stained overnight with goat anti-ACE2 (1:200, R&D Systems) in blocking buffer followed by rabbit anti-goat Alexa Fluor 488 (1:500, Invitrogen) in blocking buffer for 2 hours at room temperature, washed two times with PBS, incubated with indicated additional stains (hoechst or phalloidin), washed twice with PBS, and mounted in Prolong Antifade mounting medium 3D Alveolar and airway organoids were fixed in 4% formalin overnight, permeabilized in 0.1% Triton X-100, and blocked for 60 min in 10% normal goat serum in PBS (blocking buffer).	anti-ACE2 suggested: None
   Cells were incubated with primary antibodies overnight at 4°C in blocking buffer, washed twice with PBS, incubated with corresponding secondary antibodies (Alexa488- and 594-conjugated anti-rabbit IgG, anti-mouse IgG, or anti-mouse IgG1 (1:500; Invitrogen)) in blocking buffer for 2 hours at room temperature, washed two times with PBS, incubated with indicated additional stains (hoechst or phalloidin), washed twice with PBS, and mounted in Prolong Antifade mounting medium.	anti-rabbit IgG suggested: None anti-mouse IgG1 suggested: None Antifade mounting medium . suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Calu-3 cells were maintained in Opti-MEM I (1) + GlutaMAX (Gibco) supplemented with 10% FBS, penicillin (100 IU/mL), and streptomycin (100 IU/mL) at 37°C in a humidified CO2 incubator.	Calu-3 suggested: None
   RNA copies per ml were determined by qRT-PCR using primers targeting the E gene (52) and comparing the Ct values to a counted standard curve derived from a Bavpat-1 stock titrated on VeroE6 cells.	VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Software and Algorithms
   Sentences	Resources
   Raw deep-sequencing data are available at the NIH Short Read Archive under accession number: BioProject PRJNA722947.	BioProject suggested: (NCBI BioProject, RRID:SCR_004801)
   At 3 days postinfection light microscopy images were taken of VeroE6 infected cultures using a Zeiss Primovert microscope and analysed using ZEN software (Zeiss).	ZEN suggested: None
   Plaque assay analysis was performed using ImageQuant TL 8.2 software (GE Healthcare).	ImageQuant suggested: (ImageQuant, RRID:SCR_014246)
   Briefly, sequencing adapters from the paired-end sequencing reads were trimmed by cutadapt (54), and aligned to the corresponding genome (Bavpat-1 and B.1.1.7 isolate consensus sequence) using Bowtie2 (55) with the parameters: --no-discordant --dovetail --no-mixed -- maxins 2000.	Bowtie2 suggested: (Bowtie 2, RRID:SCR_016368)
   Variant calling was performed by VarScan2 (57) with parameters: pileup2cns --min-coverage 10 --min-reads2 2 --min-var-freq 0.01 --min- freq-for-hom 0.75 --p-value 0.05 --variants 1 using pileup data from SAMtools mpileup (58) with parameters: --excl-flags 2048 --excl-flags 256 --max-depth 50000 --min-MQ 30 --min-BQ 30.	SAMtools suggested: (SAMTOOLS, RRID:SCR_002105)
   Raw sequencing data are available at the NIH Short Read Archive under accession number: BioProject PRJNA722947.	Short Read Archive suggested: None
   Read 2 was aligned to the CRCh38 human RefSeq transcriptome, with the addition of SARS-CoV-2 (Ref-SKU: 026V-03883; MW947280) genomes, using BWA using standard settings (62).	BWA suggested: (BWA, RRID:SCR_010910)
   Differential expression analysis were performed using the DESeq2 package (63).	DESeq2 suggested: (DESeq, RRID:SCR_000154)
   Data was stitched, uploaded, shared and annotated using Omero (annotated using Omero) and PathViewer.	Omero suggested: (OMERO, RRID:SCR_002629)
   Statistical analysis: Statistical analysis was performed with the GraphPad Prism 9 software.	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

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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: 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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   Version published to 10.1101/2021.05.03.441080v1 on bioRxiv

   May 3, 2021