 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Aug 05, 2016 |
| Title |
L/L, rep1 |
| Sample type |
SRA |
| |
|
| Source name |
L/L, heart mitochondria
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6N
tissue: heart
genotype: Polrmt +/+
age: 3-4 weeks
molecule subtype: mitochondrial RNA
|
| Growth protocol |
To generate conditional Polrmt knockout mice, exon 3 was loxP-flanked (Taconic Artemis GmbH, Cologne, Germany) and excised by breeding to mice expressing cre from the muscle creatinine kinase promoter.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from crude heart mitochondria using the miRNeasy mini kit (QIAGEN) and the concentration, purity, and integrity was confirmed using a BioAnalyser.
RNA sequencing libraries were constructed using the Illumina TruSeq Sample Prep Kit. Paired end deep sequencing of the mitochondrial RNAs was performed on an Illumina MiSeq according to the manufacturer’s instructions.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
| |
|
| Data processing |
Basecalling performed with MSR: Generate FASTQ v1.0.0
Sequenced reads were aligned to the mouse genome (GRCm38) with HISAT 0.1.6 (--fr --rna-strandness RF). Reads that aligned to the mitochondria were extracted and subsequently realigned with identical parameters, but spliced alignment disabled, to reflect the un-spliced nature of the mitochondrial transcriptome and prevent the introduction of spurious splice junctions.
Gene-specific counts were summarized with featureCounts 1.3.5-p4 (-p -s 2 -Q 10) using the Ensembl 75 gene annotation for nuclear-encoded genes, and a modified annotation for mitochondrial genes. The modified annotation was based on the Ensembl 75 MT annotation, but contains merged mt-Atp8/mt-Atp6 and mt-Nd4l/mt- Nd4 annotations to reflect their bicistronic nature and accurately determine read counts.
Initially, mt-tRNAs and genes with a zero count in any sample were filtered from the count table, followed by loess and upper quartile normalization for GC-content and sequencing depth, performed with EDASeq 2.3.2.
Differential expression analysis was performed in R 3.2.0 with edgeR 3.11.2 using a GLM with tagwise dispersion estimates and the offsets generated by EDASeq.
Genome_build: GRCm38
Supplementary_files_format_and_content: Tab-delimited text files containing raw counts, normalized counts and differential expression results
|
| |
|
| Submission date |
Jun 15, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Stefan J Siira |
| Organization name |
The Kids Research Institute Australia
|
| Department |
Precision Health
|
| Lab |
Mitochondrial Medicine and Biology
|
| Street address |
15 Hospital Ave, Nedlands
|
| City |
Perth |
| State/province |
Western Australia |
| ZIP/Postal code |
6009 |
| Country |
Australia |
| |
|
| Platform ID |
GPL16417 |
| Series (1) |
| GSE83368 |
POLRMT regulates the switch between replication-primer formation and gene expression of mammalian mtDNA |
|
| Relations |
| BioSample |
SAMN05251132 |
| SRA |
SRX1846417 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2200349_S1.normalised_counts.txt.gz |
23.7 Kb |
(ftp)(http) |
TXT |
| GSM2200349_S1.raw_counts.txt.gz |
116.4 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
|
|
|
|
 |
