|
| Status |
Public on Feb 14, 2013 |
| Title |
pre-existing RNA 25hpi |
| Sample type |
SRA |
| |
|
| Source name |
NIH-3T3 fibroblasts
|
| Organism |
Mus musculus |
| Characteristics |
cell line: NIH-3T3
cell type: fibroblasts
infected with: BAC-derived MCMV (murine cytomegalovirus) Smith strain
hours past infection: 25
rna type: pre-existing RNA
|
| Treatment protocol |
Cells were seeded overnight to 80% confluence followed by infection with BAC-derived MCMV Smith strain. Infection was performed at a MOI of 10 using centrifugal enhancement (30 min, 2000 rpm) or a MOI of 0.5. The time point after centrifugation was marked as time point 0 min in all kinetics. RNA labeling was started by adding 200 µM 4-thiouridine (4sU, Sigma) to culture media for 1 h at different times of infection. At the end of labeling total cellular RNA was isolated using Trizol reagent (Invitrogen). Biotinylation and purification of 4sU-tagged RNA (newly transcribed RNA) as well as the dot blot analysis were performed as described previously (Dölken et al., RNA, 2008)
|
| Growth protocol |
Murine NIH-3T3 fibroblasts were cultured in DMEM supplemented with 5% fetal calf serum
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was subjected to WTAK library construction to generate transcriptomic fragment libraries (50 bp, SOLiD™ (Life Technologies, Foster City, CA, USA) Total RNA-Seq Kit V3) that preserve strandedness information of the reads. Molecular barcoding was used in order to pool several libraries in a single sequencing reaction according to the manunfacturer´s protocol. Sequencing was performed using the SOLiDTM 3 system (Life Technologies). Potential sequencing errors were corrected using the SOLiD Accuracy Enhancement Tool (solidsoftwaretools.com/gt/project/saet).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
AB SOLiD System 3.0 |
| |
|
| Description |
894
|
| Data processing |
ALIGNMENT: Reads were aligned in a 4-step process using the Bowtie alignment program. First, all reads were aligned to mouse transcripts (Ensembl version 63). Remaining unaligned reads were aligned to the mouse genome (mm9, NCBI Build 37). Then, remaining reads were aligned to MCMV coding sequences and finally to the full MCMV genomic sequence. Reads with ambiguous base calls, non-unique alignment positions or more than 4 mismatches were discarded.
WIG: Wiggle files were created for the virus genome using all aligned reads.
RPKM: RPKM values were calculated for MCMV genes. Therefore, all reads aligning to a gene were summed and normalized. The length of the sequence covered by the gene was used for length normalization. The total number of reads aligned to mouse exons (Ensembl version 63) and virus genes was used for sample normalization.
Genome Build:
894_+_strand.wig: BAC-derived MCMV Smith strain
894_-_strand.wig: BAC-derived MCMV Smith strain
894.rpkm: BAC-derived MCMV Smith strain
|
| |
|
| Submission date |
Feb 15, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Lukas Windhager |
| E-mail(s) |
Lukas.Windhager@bio.ifi.lmu.de
|
| Phone |
00498921804055
|
| Fax |
0049892180994055
|
| Organization name |
Ludwig-Maximilians-Universität Munich
|
| Department |
Institut für Informatik
|
| Lab |
LFE Bioinformatik
|
| Street address |
Amalienstr. 17
|
| City |
Munich |
| ZIP/Postal code |
80333 |
| Country |
Germany |
| |
|
| Platform ID |
GPL9318 |
| Series (1) |
| GSE35833 |
Real-time transcriptional profiling of cellular and viral gene expression during lytic cytomegalovirus infection |
|
| Relations |
| SRA |
SRX120045 |
| BioSample |
SAMN00790448 |
