|
| Status |
Public on Oct 03, 2023 |
| Title |
WT_mock_4_6hpi_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
A549-ACE2
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: A549-ACE2
genotype: wild type
cell type: mock infected
treatment: 2h 4sU
|
| Treatment protocol |
A549-ACE2 SND1 knockout cells, control cells, or parental A549-ACE2 wild-type cells were infected with SARS-CoV-2 at MOI 3 PFU/cell. For each time point 4-Thiouridine was added to the cell culture medium to a final concentration of 200 μM and cells were returned to the incubator for 2 h.
|
| Growth protocol |
We maintained A549-ACE2 cells in DMEM media (Thermo Fisher Scientific) supplemented with 10% heat-inactivated FBS (Thermo Fisher Scientific), and 100 units/ml streptomycin and 100 mg/ml penicillin. Cells were grown at 37°C and 5% CO2 atmosphere.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested in Trizol and RNA was extracted using the Direct-zol RNA microprep kit (Zymo Research) including DNase digest. From each sample, 2 μg of RNA were subjected to alkylation by incubating with 10 mM Iodoacetamide in PBS with 50% DMSO at 50°C for 15 min as previously described (Herzog, V.A., et al. Thiol-linked alkylation of RNA to assess expression dynamics. Nat Methods 14, 1198–1204 (2017). After adding an equal volume of H2O to the sample, the RNA was purified using the RNA Clean and Concentrator-5 kit (Zymo Research).
Approximately 800 ng of alkylated RNA was depleted of ribosomal RNA using the NEBNext rRNA Depletion Kit v2 (NEB) according to the manufacturer’s instructions. RNA was heat fragmented at 91°C for 3 min in 10 mM Tris-HCl pH 8.0, 5 mM MgCl2, 100 mM KCl, 0.02% Triton X-100 and 0.1 mg/mL BSA. RNA was dephosphorylated using 1.75 U FastAP Thermosensitive Alkaline Phosphatase for 10 min at 37°C , and end-repaired using 15 U T4 PNK for 20 min at 37°C. Both reactions were performed in the presence of 10 U murine RNase inhibitor. RNA was cleaned up using Dynabeads MyOne Silane (Thermo Fisher Scientific). For this, 10 μl beads per sample were washed twice with RLT buffer (Qiagen) and beads were added to the RNA in 3x sample volumes RLT buffer. To bind the RNA to the beads, 4.5x volumes isopropanol were added and samples were incubated 5 min at room temperature. Beads were washed twice with 70% EtOH and RNA was eluted in 8 μl H2O. cDNA libraries were constructed from the recovered RNA as described in the Methods section of the accompanying paper.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
SLAM-seq was performed in duplicates to identify newly synthesized RNA and total RNA using GRAND-SLAM. Sequencing adapters were trimmed using Cutadapt (version 3.4). Next, bowtie2 (version 2.3.0) (Langmead and Salzberg, 2012) with default parameters was used to discard reads mapping to rRNA (Genbank identifier U13369.1) and to verify the absence of mycoplasma contamination. STAR version 2.5.3a (REF) was used to map all remaining reads with length at least 18 nt against a combined index of the human genome (hg38/Ensembl version 90) and the SARS-CoV-2 genome (NC_045512) using STAR (version 2.5.3a) with the parameters –outFilterMismatchNmax 20 –outFilterScoreMinOverLread 0.4 –outFilterMatchNminOverLread 0.4 –alignEndsType Extend5pOfReads12 –outSAMattributes nM MD NH. We used GRAND-SLAM (version 2.0.6) to estimate the new-to-total RNA ratios. New RNA was computed by multiplying total RNA with the maximum a posteriori estimate of the new-to-total RNA ratio. Log2 fold-changes were estimated using PsiLFC (Erhard, 2018). Normalization factors were computed from total RNA such that the median log2 fold-change was 0 and applied to both total and new RNA.
Assembly: hg38 and NC_045512
Supplementary files format and content: comma separated file; log2 fold-changes with respect to corresponding WT sample
|
| |
|
| Submission date |
Nov 15, 2022 |
| Last update date |
Oct 03, 2023 |
| Contact name |
Alexander Gabel |
| E-mail(s) |
alexander.gabel@helmholtz-hiri.de
|
| Organization name |
Helmholtz Institute for RNA-based Infection Research
|
| Lab |
LncRNA and Infection Biology (LRIB)
|
| Street address |
Josef-Schneider-Str. 2 / D15
|
| City |
Würzburg |
| ZIP/Postal code |
97080 |
| Country |
Germany |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE217429 |
SND1 binds SARS-CoV-2 negative-sense RNA and promotes viral RNA synthesis through NSP9 |
|
| Relations |
| BioSample |
SAMN31744310 |
| SRA |
SRX18276615 |
