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Sample GSM2200354 Query DataSets for GSM2200354
Status Public on Aug 05, 2016
Title L/L, cre rep3
Sample type SRA
Source name L/L cre, 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 Harry Perkins Institute of Medical Research
Department Molecular medicine
Lab Mitochondrial medicine and biology
Street address 6 Verdun St, 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
BioSample SAMN05251137
SRA SRX1846422

Supplementary file Size Download File type/resource
GSM2200354_S6.normalised_counts.txt.gz 24.2 Kb (ftp)(http) TXT
GSM2200354_S6.raw_counts.txt.gz 119.8 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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