|
| Status |
Public on Aug 04, 2016 |
| Title |
WT-522 |
| Sample type |
SRA |
| |
|
| Source name |
RNASeq_WT_rep2
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Heart
age: 11 weeks
genotype: Mrpp3^loxP/loxP
strain: C57BL/6N
molecule: total cellular RNA from heart
|
| Growth protocol |
Mice were housed in standard cages (45 cm × 29 cm × 12 cm) under a 12-h light/dark schedule (lights on 7 am to 7 pm) in controlled environmental conditions of 22 ± 2 °C and 50 + 10% relative humidity and fed a normal chow diet (Rat & Mouse Chow, Specialty Foods, Glen Forrest, Western Australia) and water were provided ad libitum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from total hearts or heart mitochondria using the miRNeasy Mini kit (Qiagen) incorporating an on-column RNase-free DNase digestion to remove all DNA.
Total RNA libraries were prepared according to the Illumina TruSeq protocol, using random hexamer primers for cDNA generation and the Ribo-Zero rRNA removal kit for cytoplasmic RNA deptetion. Mitochondrial RNA was prepared according to the PARE protocol as described by German et al. (Nature Protocols, Vol.4 No.3, 2009).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Basecalling was performed by HiSeq Control Software
Quality and adapter trimming was performed with Flexbar v2.5
To analyse the nuclear transcriptome:
Reads were aligned to the mouse genome using HiSat v.0.1.6-beta.
Guided transcriptome assembly (GRCm38.81) was done using StringTie v.1.1.0.
Cufflinks v.2.2.1 was then used for analysis of differential expression. Briefly, cuffmerge was used to merge all assemblies with the reference assembly as template, and cuffquant followed by cuffdiff (--dispersion-method per-condition) for quantification and differential analysis.
genome build: GRCm38
Tab-delimited txt containing FPKM values for each sample calculated by CuffDiff
To analyse the mitochondrial trancriptome:
Sequenced reads were initially aligned to the mouse genome with HISAT2 v2.0.1-beta (--very-sensitive --fr --rna-strandness RF --phred33 --no-mixed --no-discordant). The read IDs of all mitochondrial primary alignments were extracted with SAMtools v1.3, and used to filter the original alignments into nuclear and mitochondrial alignment files with Picard v1.23. The mitochondrial alignments were converted back to their original read sequence in fastq format with Picard, and realigned to the mitochondrial genome as before with the additional parameter --no-spliced-alignment.
Primary mitochondrial alignments were extracted and separated according to their template strand-of-origin with SAMtools and converted to template strand-specific fragment BED files with BEDtools and awk.
Per-base depth of strand-specific fragment BED files were generated with BEDtools normalized by the total number of properly mapped pairs per million across the whole genome (nuclear and mitochondrial) and converted to bedGraph format.
genome build: mm10
Strand-specific fragment coverage for each sample in bedGraph format, in reads per million (RPM)
|
| |
|
| Submission date |
Jun 17, 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 |
GPL13112 |
| Series (1) |
| GSE83471 |
Hierarchical RNA processing is rate limiting for mitochondrial ribosome assembly |
|
| Relations |
| BioSample |
SAMN05257263 |
| SRA |
SRX1852588 |
