|
| Status |
Public on Nov 19, 2013 |
| Title |
sRNAs from siRNaseL transfected and SINV-infected HEK293 cells |
| Sample type |
SRA |
| |
|
| Source name |
siRNaseL transfected and SINV-infected HEK293 cells
|
| Organism |
Homo sapiens |
| Characteristics |
host cell line: HEK293
transfection of host cell line: siRNaseL
viral infection of host cell line: Sindbis virus
viral infection conditions: 16 hpi, MOI 0.01
|
| Growth protocol |
HEK293 and VERO 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. Cells were infected with SINV at MOI 0.01. Samples were harvested at 16 hours post-infection (hpi).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from non-infected and infected cells using Tri-reagent (Ambion) as per manufacturer’s instructions.
Small RNA libraries were prepared from 10 or 20 μg of total RNA as previously described (Pfeffer, S. 2007. Identification of virally encoded microRNAs. Methods Enzymol. 427:51-63), except that PCR products were not concatemerized but directly sent for large-scale sequencing. Libraries from samples 5 to 10 were generated using the same protocol except that degenerate 5' and 3' adapters were used to limit ligation biases.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Small RNA profiling
Sample 6
siRNaseL transfection was performed before infection and total RNA extraction
|
| Data processing |
Base calling were performed using CASAVA v.1.6.0 (samples 1 to 4) and v.1.8.2 (samples 5 to 10)
Sequencing reads were preprocessed using the Dustmasker program and FASTX-Toolkit to filter out low complexity sequences and to remove instances of the 3' adaptor respectively. If needed, degenerate bases incorporated during the new library preparation protocol were also trimmed. For siCtl and siRNAseL libraries, we also eliminated reads corresponding to the siRNA sequences.
Remaining reads of at least 15 nt in length were then mapped simultaneously to the human and SINV genomes using Bowtie 0.12.7. Up to 2 mismatches in total with no more than 1 mismatch in the first 15 nucleotides of each read were permitted. In addition, only alignments from the lowest mismatch-stratum were recorded and reads that could map to more than 50 loci were discarded.
For each source 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 distribution along the viral genome.
Genome_build: GRCh37/hg19 + NC_001547.1
Processed data files format and content: .bed is an alignment file of only the viral reads for each library (Reference, Begin [0-based offset], End [1-based offset], Name [=raw readcount], Score [=raw readcount], Strand)
Processed data files format and content: Summary.txt (linked as a supplementary file on the Series record) is a tab-delimited file with a detailed description of all the alignments + sequences obtained in the 10 libraries (Length, Sequence, Raw readcount in each of the 10 libraries, Reference, Begin [1-based offset], End [1-based offset], Strand, Mismatch number, Mismatch description [position_in_the_read : base_in_the_reference > base_in_the_read])
|
| |
|
| Submission date |
Jul 12, 2013 |
| 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 |
GPL16791 |
| Series (1) |
| GSE48831 |
Viral small RNAs in Sindbis virus-infected mammalian cells |
|
| Relations |
| SRA |
SRX322092 |
| BioSample |
SAMN02231987 |
