Multi-color super-resolution imaging to study human coronavirus RNA during cellular infection
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SciScore for 10.1101/2021.06.09.447760: (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 simultaneous staining of gRNA and dsRNA, cells were incubated with 100 μL Hybridization Buffer containing 2 μL 12.5 μM AF647-labled RNA FISH probes and 1:1000 anti-dsRNA antibodies ( anti-dsRNAsuggested: NoneThen cells were incubated with 1:20000 CF™ 568 Donkey anti-mouse antibody for 30 min at 37°C, washed with Wash Buffer A containing DAPI for 30 min at 37°C in the dark, and stored in Wash Buffer B for imaging. anti-mousesuggested: NoneExperimental Models: Cell Lines Sentences Resources Lentivirus production: To produce lentivirus, HEK293T cells were transiently transfected with the lentiviral … SciScore for 10.1101/2021.06.09.447760: (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 simultaneous staining of gRNA and dsRNA, cells were incubated with 100 μL Hybridization Buffer containing 2 μL 12.5 μM AF647-labled RNA FISH probes and 1:1000 anti-dsRNA antibodies ( anti-dsRNAsuggested: NoneThen cells were incubated with 1:20000 CF™ 568 Donkey anti-mouse antibody for 30 min at 37°C, washed with Wash Buffer A containing DAPI for 30 min at 37°C in the dark, and stored in Wash Buffer B for imaging. anti-mousesuggested: NoneExperimental Models: Cell Lines Sentences Resources Lentivirus production: To produce lentivirus, HEK293T cells were transiently transfected with the lentiviral plasmid, and packaging plasmids pCMV-dR8.91 and PMD2.G. HEK293Tsuggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)229E production: Human coronavirus 229E (Cat# VR-740, ATCC) is amplified once by inoculating one T75 flask of fully confluency MRC-5 cells. MRC-5suggested: ICLC Cat# HL95001, RRID:CVCL_0440)Infection of the cells by 229E: MRC-5 cells or MRC-5-ER-GFP cells were seeded at 4-4.5 × 104 cells per well into poly-D-lysine-treated 8-well μ-slides (Cat# 80826, IBIDI) one day before infection. MRC-5-ER-GFPsuggested: NoneConfocal image analysis: To quantify the number of infected MRC5 cells at different stages of the infection in Fig 4C and Fig 6A, we counted cells with both gRNA and dsRNA staining as infected cells in FIJI. MRC5suggested: NoneRecombinant DNA Sentences Resources Lentivirus production: To produce lentivirus, HEK293T cells were transiently transfected with the lentiviral plasmid, and packaging plasmids pCMV-dR8.91 and PMD2.G. pCMV-dR8.91suggested: NoneGeneration of stable cell lines: MRC-5-ER-GFP stable cells were constructed by transducing the MRC-5 cells with lentivirus expressing pLV-ER-GFP (Cat# 80069, Addgene, a gift from Pantelis Tsoulfas), followed by sorting for cells expressing GFP using a SONY SH800S sorter. pLV-ER-GFPsuggested: NoneSoftware and Algorithms Sentences Resources To quantify the number of dsRNA puncta in each cell in Fig 4D and Fig 6B, the Trackmate plugin in FIJI was used. FIJIsuggested: (Fiji, RRID:SCR_002285)SR data analysis: We processed approximately 40000 frames of single-molecule images for 2D Gaussian fitting by ThunderSTORM (ImageJ). ImageJsuggested: (ImageJ, RRID:SCR_003070)Clustering analysis: To calculate the sizes of gRNA and dsRNA clusters as well as the number of molecules within each cluster, we used Voronoi tessellation on the single molecules from the previous step using a custom MATLAB script. MATLABsuggested: (MATLAB, RRID:SCR_001622)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).
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.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
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.
Results from scite Reference Check: We found no unreliable references.
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