Data on integrative vectors for the regulated expression of SARS-CoV-2 proteins implicated in RNA metabolism

This figshare record contains 4 zipped folders: C terminal constructs Sanger sequencing.zip, Parental vectors Sanger sequencing.zip, Untagged and N terminal constructs Sanger sequencing.zip and Licor Imaging.zip.

The Sanger sequencing folders contain data files in .dna, .ab1 and .seq file formats.

The Licor Imaging folder contains data files in .tif, .txt and .jpg file format.

The data record also includes 3 files in .docx file format, and 4 tables in .xlsx file format.

The document Primary-Data-Index.docx includes information about what is contained in each folder and data file.

The data file names have the following structure: sample name - primer - sequencing name.

Below is a short description of the contents of each of the folders, the .docx files and the .xlsx files:

1. Cloning FH and HF containing-vectors.docx: Protocol and lab notes for cloning and screening colonies for the parental tagging vectors

2. C terminal tagged and eGFP construct cloning.docx: Protocol and original gel images for cloning and screening colonies for C terminally tagged constructs. Untagged, FH and HF eGFP constructs gel images are also included in this file.

3. Untagged and N-terminal tagged construct cloning.docx: Protocol and original gel images for cloning and screening colonies for C terminally tagged constructs.

4. Parental vectors Sanger sequencing.zip: Original Sanger sequencing files for confirming the parental tagging vectors.

5. C terminal constructs Sanger sequencing.zip: Original Sanger sequencing files for confirming C terminally tagged constructs, provided embedded in SnapGene files as well as original files

6. Untagged and N terminal constructs Sanger sequencing.zip: Original Sanger sequencing files confirming untagged and N-terminally tagged constructs.

7. Licor imaging.zip: Original scans of the western blots performed.

Table 3.xlx includes data on the quantification of protein abundance, following different induction time courses.

Table 4.xlsx includes the mass spectrometry data for the expression of proteins tagged at the N-terminus.

Table 5.xlsx includes the oligonucleotide sequences used in this study.

Table 6.xlsx includes the sequences of fusion protein open reading frames (ORFs).

Study aims and methodology:

SARS-CoV-2 is a large positive-sense, single-stranded RNA virus that encodes four structural proteins, several accessory proteins, and sixteen nonstructural proteins (nsp1-16). The latter are mainly engaged in enzymatic activities important for translation and replication of the RNA genome. In addition, nonstructural proteins also target host RNA metabolism in order to manipulate cellular gene expression or facilitate immune evasion. Understanding these pathways in greater detail will be an important step in the development of antiviral therapies.

In this study, the authors generated a series of synthetic, codon-optimized constructs for 14 different viral proteins that are expected to interact with RNA or RNA binding proteins. To remove the need for error-prone PCR steps, they devised a cloning scheme in which a single synthetic construct could be used to generate untagged, N-, or C-terminally tagged versions of the protein.

Using these vectors, the authors generated and tested a series of human cell lines with the viral open reading frames (ORFs) stably integrated, under the control of an inducible Tet-On promoter.

For details on the methodology, please read the related article.

Software needed to access data: Datasets in .dna and .ab1 file format can be accessed using the SnapGene software.