GSE74583
GSE GEONext Generation Sequencing Investigation of altered transcripts in presence of dominant-negative transcription factor
Relations
Summary
Purpose:The goals of this study was to determine alterations in expression levels of transcripts downstream of a dominant-negative transcription factor. Quantitative reverse transcription polymerase chain reaction (qRTâPCR) methods was used to confirm the altered expression of targets. Methods: Striatal mRNA profiles of 11-month-old wild-type (WT) and Nestin-Cre X PPAR delta E411P mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with TopHat followed by Cufflinks. qRTâPCR validation was performed using TaqMan and SYBR Green assays. Western blots, and immunofluorescence was also used to confirm if altered mRNA levels translated to changes at the protein level. Results: Using data analysis workflow, we mapped sequence reads for each sample to the mouse genome (build mm9) and identified transcripts in the striatum of WT and PPARdelta E411P mice. Conclusions: Our study found multiple transcripts altered in the striatum of the Nestin-Cre x PPAR delta E411P mice as compared to WT striatum, as generated by RNA-SEQ in biologic replicates.
Overall Design
Striatal mRNA profiles of 11-month-old wild type (WT) and Nestin-Cre X PPAR delta E411P mice were generated by deep sequencing, in triplicate, using Illumina HiSeq2000.
Analysis (5 steps)
View Data Processing- Illumina Casava1.7 software used for basecalling.
- Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm8 whole genome using bowtie v0.12.2 with parameters -q -p 4 -e 100 -y -a -m 10 --best --strata
- Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009.
- In short, exons from all isoforms of a gene were merged to create one meta-transcript.
- The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Supplementary Files (1)
GEO Samples (6)
Dataset Citations (1)
SRA Experiments (6) and Runs (6)
Total: 5206 MBSample attributes
Original files (1)
Runs (1)
| Run | Spots | Bases | Size (MB) | Files | Link |
|---|---|---|---|---|---|
| SRR2889691 | 26390582 | 1293138518 | 838.51 | FA1395.fq.gz, SRR2889691, SRR2889691.sralite | SRA |
Sample attributes
Original files (1)
Runs (1)
| Run | Spots | Bases | Size (MB) | Files | Link |
|---|---|---|---|---|---|
| SRR2889692 | 24889960 | 1219608040 | 790.03 | FA1411.fq.gz, SRR2889692, SRR2889692.sralite | SRA |
Sample attributes
Original files (1)
Runs (1)
| Run | Spots | Bases | Size (MB) | Files | Link |
|---|---|---|---|---|---|
| SRR2889693 | 29394956 | 1440352844 | 935.84 | FA1412.fq.gz, SRR2889693, SRR2889693.sralite | SRA |
Sample attributes
Original files (1)
Runs (1)
| Run | Spots | Bases | Size (MB) | Files | Link |
|---|---|---|---|---|---|
| SRR2889694 | 26904961 | 1318343089 | 852.59 | FA1376.fq.gz, SRR2889694, SRR2889694.sralite | SRA |
Sample attributes
Original files (1)
Runs (1)
| Run | Spots | Bases | Size (MB) | Files | Link |
|---|---|---|---|---|---|
| SRR2889695 | 30824198 | 1510385702 | 979.75 | FA1377.fq.gz, SRR2889695, SRR2889695.sralite | SRA |
Sample attributes
Original files (1)
Runs (1)
| Run | Spots | Bases | Size (MB) | Files | Link |
|---|---|---|---|---|---|
| SRR2889696 | 25234936 | 1236511864 | 809.0 | FA1397.fq.gz, SRR2889696, SRR2889696.sralite | SRA |