|
| Status |
Public on Aug 04, 2016 |
| Title |
KO-76 |
| Sample type |
SRA |
| |
|
| Source name |
PARE_KO_rep1
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Heart
age: 11 weeks
genotype: Mrpp3^loxP/loxP, +/Ckmm
strain: C57BL/6N
molecule: total mitochondrial 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 |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Mitochondria were isolated from homogenized hearts and isolated by differential centrifugation as described previously (Lagouge et al., 2015; Mourier et al., 2014), with some modifications. Hearts were cut and washed three times with ice cold PBS, and once with mitochondrial isolation buffer (MIB) containing 310 mM sucrose, 10 mM Tris-HCl and 0.05 % BSA (w/v) by centrifugation at 4,500 g for 1 min at 4°C. Heart pieces were homogenized in 5 ml of fresh MIB using a Potter S pestle (Sartorius). The homogenate was centrifuged at 1,000 g for 10 min at 4°C and the supernatant was centrifuged at 4,500 g for 15 min at 4°C to isolate mitochondria. Crude mitochondrial pellets were suspended in MIB supplemented with 1x Complete EDTA-free protease inhibitor cocktail (Roche).
|
| Data processing |
Library strategy: PARE
Basecalling was performed by NextSeq Control Software
The 5 bp adapter sequence was trimmed and paired-end reads were merged with CLC Genomics Workbench (Qiagen) if the read overlap exceeded 95% identity.
Merged reads were aligned to the mouse mitochondrial genome (chrM) with Bowtie2 using default parameters
Strand-specific depth of read coverage at the 5' terminal position of aligned reads was calculated with BEDTools (-d -5 -strand [+/-])
Read depth was normalised using the trimmed mean of M values (TMM) method from the edgeR package
genome build: mm10
Strand-specific 5' end coverage for each sample in bedGraph format, normalised by TMM (edgeR)
|
| |
|
| 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 |
GPL19057 |
| Series (1) |
| GSE83471 |
Hierarchical RNA processing is rate limiting for mitochondrial ribosome assembly |
|
| Relations |
| BioSample |
SAMN05257271 |
| SRA |
SRX1852596 |
