|
| Status |
Public on Dec 28, 2015 |
| Title |
sRNAs from sihDicer transfected and SINV-infected HEK293 cells expressing DAR (=Dicer-2 and R2D2), 6 hpi |
| Sample type |
SRA |
| |
|
| Source name |
HEK293 cell line
|
| Organisms |
Homo sapiens; Sindbis virus |
| Characteristics |
cell line: HEK293
construct: pFlag-HA-Dicer2-Puro + pIRES-V5-R2D2/myc-Ago2-Neo
growth protocol: HEK293 cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere of 5% CO2 at 37°C.
viral infection condition: 6 hours post-infection, SINV MOI 0.01
extract protocol: Total RNA was extracted from infected cells using TRI-Reagent (Ambion, AM9738)
|
| Growth protocol |
(Included as an additional column of the SAMPLES, see section above)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
(Included as an additional column of the SAMPLES, see section above)
Small RNA libraries were prepared from 10 μg of total RNA as previously described (Contrant et al. Importance of the RNA secondary structure for the relative accumulation of clustered viral miRNAs. Nucleic Acids Res. 2014;42(12):7981-96)
|
| |
|
| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Small RNA
sihDicer transfection was performed 2 days before SINV infection
|
| Data processing |
Image analysis and base calling were performed using RTA 1.17.21.3 and CASAVA 1.8.2.
Sequencing reads were preprocessed using the Dustmasker program and FASTX-Toolkit to filter out low complexity reads and remove instances of the 3' adapter. Degenerate bases incorporated during the library preparation protocol were then trimmed and remaining reads were further size-selected by keeping only the 18- to 32-nt long ones. In the case of human libraries, exogenous siRNA sequences were also excluded before further analysis.
Preprocessed reads were then mapped simultaneously to the host (D. melanogaster or H. sapiens) and the pathogen (SINV) genomes using Bowtie 1.0.0. In the case of fly libraries, small RNA reads were also mapped to the white-inverted repeats, wIR, transgene, as previously shown (Marques et al., 2010). Initially, only alignments of reads with 0 mismatch were recorded and reads that could map to more than 50 loci were discarded.
For each library, small RNAs deriving solely from SINV, in either sense or antisense orientation, were computationally extracted and profiled based on their length distribution and their coverage along the viral genome. The same approach was used to deal with reads deriving from the wIR transgene in fly libraries.
In order to establish the host miRNA expression profiling, host-specific reads presenting with up to 2 mismatches in total with no more than 1 mismatch in their first 15 nucleotides were taken into account.
Fly or human miRNAs (miRBase 20) were annotated in each library using BEDTools 2.16.2. During the quantification process, multiple mapped reads were weighted by the number of mapping sites in miRNAs.
To visually explore miRNA expression profiles in each library set, we produced heatmaps showing the expression data of the 100 most highly expressed miRs, after regularized log transformation (rlog2), as recommended in the DESeq2 package.
Genome_build: Dmel r5.54 + NC_001547.1 (samples 1 to 3) and GRCh37/hg19 + NC_001547.1 (samples 4 to 11).
Supplementary_files_format_and_content: Alignment files of the only viral reads are provided in BED format (*-onlyviral-alignments.bed) as well as the annotation files of the 100 most abundant miRNAs in tab-delimited format (*-top100-cellular-miRs-annot.txt: Column 1: miR, Column 2: rlog2).
|
| |
|
| Submission date |
Nov 03, 2014 |
| Last update date |
May 15, 2019 |
| 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 |
GPL19381 |
| Series (1) |
| GSE62934 |
Cross-species comparative analysis of Dicer proteins during Sindbis virus infection |
|
| Relations |
| BioSample |
SAMN03160310 |
| SRA |
SRX750166 |
