Gene Expression
Nebulas
Gene Expression NebulasSummary: COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which infected >200 million people resulting in >4 million deaths. However, temporal landscape of the SARS-CoV-2 translatome and its impact on the human genome remain unexplored. Here, we report a high-resolution atlas of the translatome and transcriptome of SARS-CoV-2 for various time points after infecting human cells. Intriguingly, substantial amount of SARS-CoV-2 translation initiates at a novel translation initiation site (TIS) located in the leader sequence, termed TIS-L. Since TIS-L is included in all the genomic and subgenomic RNAs, the SARS-CoV-2 translatome may be regulated by a sophisticated interplay between TIS-L and downstream TISs. TIS-L functions as a strong translation enhancer for ORF S, and as translation suppressors for most of the other ORFs. Our global temporal atlas provides compelling insight into unique regulation of the SARS-CoV-2 translatome and helps comprehensively evaluate its impact on the human genome.
Overall Design: mRNA-seq, RPF-seq, QTI-seq, and sRNA-seq experiments at uninfected state and at 0, 1, 2, 4, 12, 16, 24, 36, and 48 hpi upon SARS-CoV-2 infection in human Calu-3 cell lines.
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| Growth Protocol: | Calu-3 cells were cultured in DMEM with 10% FBS and 1% penicillin/streptomycin |
| Treatment Protocol: | Prior to lysis, cells were seeded in 100mm dishes, and treated cells were infected with SARS-CoV-2 (MOI = 0.1). At 48 hpi, both mock and treated dishes for RPF, QTI, and mRNA-seq were treated with either 100 μg/ml cycloheximide (CHX) or 2 µg/ml harringtonine (Harr) for 10 min. |
| Extract Protocol: | For sRNA-seq, RNA was extracted using Trizol. For rest, cells were washed with PBS then lysed with lysis buffer (10 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 100 mM KCl, 1% Triton X-100, 100 μg/ml CHX, 1 mM dithiothreitol, 0.2 U/μl RiboLock RNase inhibitor, and 1 × EDTA-free protease inhibitor cocktail). For RPF-seq and QTI-seq, lysates were treated with RNase I and ribosome protected fragments were isolated using illustra MicroSpin S-400 HR Columns (GE Healthcare). |
| Library Construction Protocol: | All of the libraries (mRNA-seq, RPF-seq, QTI-seq, and sRNA-seq) were constructed following the Illumina Truseq small RNA library preparation protocol (TruSeq Small RNA Library Prep Guide, Part # 15004197 Rev. G) |
| Molecule Type: | rRNA- RNA |
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| Library Layout: | SINGLE |
| Library Strand: | Reverse |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 2500 |
| Strand-Specific: | Specific |