|
| Status |
Public on Jan 26, 2024 |
| Title |
Dicer WT, SINV-GFP MOI 2 12 hpi, rep3 |
| Sample type |
SRA |
| |
|
| Source name |
HEK293T
|
| Organisms |
Homo sapiens; Sindbis virus |
| Characteristics |
cell line: HEK293T
cell type: Human embryonic kidney
genotype: NoDice FHA:DICER WT #4
viral infection_conditions: MOI 2, 12 hpi
|
| Treatment protocol |
Cells were infected with a modified version of Sindbis virus expressing GFP (SINV-GFP) at an MOI of 2 for 12 h or 0.02 for 24 h. Cells were maintained in Dulbecco’s modified Eagle medium supplemented with 10 % fetal bovine serum in a humidified atmosphere with 5 % CO2 at 37°C.
|
| Growth protocol |
NoDice FHA:DICER WT #4 or N1 #6 cells were plated in P100mm petri dishes at 5 000 000 cells per dish and infected the following day with SINV-GFP at an MOI of 2 for 12 h or 0.02 for 24 h. Cells were maintained in Dulbecco’s modified Eagle medium supplemented with 10 % fetal bovine serum in a humidified atmosphere with 5 % CO2 at 37°C.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Cells were collected in Trizol reagent (Invitrogen, Thermo Fisher Scientific) and RNAs were isolated according to the manufacturer’s instructions.
ssRNA-seq (stranded RNA-seq): RNA-Seq libraries were generated according to the manufacturer’s instructions from 250 ng of total RNA using the Illumina Stranded mRNA Prep, Ligation kit (Reference Guide - PN 1000000124518) and IDT for Illumina RNA UD Indexes Ligation (Illumina, San Diego, USA). Briefly, Oligo(dT) magnetic beads were used to purify and capture the mRNA molecules containing polyA tails.The purified mRNA were then fragmented at 94°C for 2 min and copied into first strand complementary DNA (cDNA) using reverse transcriptase and random primers. Second strand cDNA synthesis further generated blunt-ended double-stranded cDNA and incorporated dUTP in place of dTTP to achieve strand specificity by quenching the second strand during amplification. Following A-tailing of DNA fragments and ligation of pre-index anchors, PCR amplification was used to add indexes and primer sequences and to enrich DNA libraries (30 sec at 98°C; [10 sec at 98°C, 30 sec at 60°C, 30 sec at 72°C] x 12 cycles; 5 min at 72°C). Surplus PCR primers were further removed by purification using SPRIselect beads (Beckman-Coulter, Villepinte, France) and the final libraries were checked for quality and quantified using capillary electrophoresis.
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| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
Homo sapiens and Sindbis-GFP virus
|
| Data processing |
Image analysis and base calling were performed using RTA 2.7.7 and bcl2fastq 2.20.0.422.
RNA-seq data were processed and analyzed using the following workflow.
The first 5'-end base of each read, a T-overhang added during the library preparation with the Illumina Stranded mRNA protocol, was first trimmed, before using Skewer v0.2.2 in paired-end mode for average quality read filtering and adapter trimming (command: skewer -Q 25 -x CTGTCTCTTATACACATCT -y CTGTCTCTTATACACATCT -l 31 -m pe -t 2 -o <name> --quiet <name>_R1.fq <name>_R2.fq).
Sample quality checks were performed before and after these preprocessing steps using FastQC v0.11.8.
Then, human full-length protein-coding transcripts (GENCODE Human (GRCh38.p13) release 41) and SINV-GFP both genomic and subgenomic transcripts (private sequences derived from NC_001547.1) were quantified using Salmon v1.10.0 in mapping-based mode with the selective alignment algorithm and a decoy-aware transcriptome (command: salmon quant -p 6 -i index/gencode41.hg38.sinv.decoys_index --libType A --seqBias --gcBias --numBootstraps 30 -1 data/<name>_preprocessed_R1.fq.gz -2 data/<name>_preprocessed_R2.fq.gz -o salmon-quants/<name>).
Transcript-level abundance and count estimates thus obtained were next imported into R and further analyzed with the tximport and DESeq2 packages. Briefly, original transcript-level counts were summarized to gene-level estimated counts and an offset was produced from transcript-level abundance estimates to correct for changes to the average transcript length across samples. Differential expression analyses between tested conditions were then conducted at the gene-level, and statistical significance was defined with an adjusted p-value < 0.05 and an absolute log2 fold change > 1 thresholds.
Finally, gene set enrichment analysis (GSEA) studies were performed using the GSEA_4.3.2 Mac application, as made available by the Broad Institute and the University of California, San Diego (https://www.gsea-msigdb.org/gsea/index.jsp). These analyses were conducted on the DESeq2 normalized counts of each comparison dataset against the Hallmark Gene Sets (v2023.1) provided by the Molecular Signatures Database (MSigDB), and by using the Human_Ensembl_Gene_ID_MSigDB.v2023.1.Hs.chip annotation file and all other parameters by default, except the permutation type which was set to gene_set
Assembly: GRCh38.p13 + private sequence derived from NC_001547.1
Supplementary files format and content: salmon_quants.txt: tab-delimited text file with Salmon quantification of studied transcripts in each sample
Supplementary files format and content: deseq2_normcounts.txt: tab-delimited text file with DESeq2 normalized gene counts in each sample
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| |
|
| Submission date |
Aug 29, 2023 |
| Last update date |
Jan 26, 2024 |
| Contact name |
Sébastien Pfeffer |
| Organization name |
CNRS - Institut de Biologie Moléculaire et Cellulaire (IBMC)
|
| Department |
UPR 9002 - Architecture et Réactivité de l'ARN
|
| Lab |
RNA regulation in viral infections
|
| Street address |
2 allée Konrad Roentgen
|
| City |
Strasbourg |
| ZIP/Postal code |
67084 |
| Country |
France |
| |
|
| Platform ID |
GPL33716 |
| Series (2) |
| GSE241796 |
The helicase domain of human Dicer prevents RNAi-independent activation of antiviral and inflammatory pathways |
| GSE241798 |
The helicase domain of human Dicer prevents RNAi-independent activation of antiviral and inflammatory pathways |
|
| Relations |
| BioSample |
SAMN37179211 |
| SRA |
SRX21500029 |
