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| Status |
Public on Nov 21, 2017 |
| Title |
LRPPRC_KO_RNase_T1 |
| Sample type |
SRA |
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| Source name |
Heart mitochondria
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6N
tissue: heart
cell compartment: mitochondria
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| Growth protocol |
Knockout transgenic mice on a C57BL/6N background 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 and water were provided ad libitum.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Mitochondria were isolated from homogenized hearts and isolated by differential centrifugation as described previously (Lagouge et al., PLoS Genetics, 2015; Mourier et al., Human Molecular Genetics, 2014), with some modifications. Hearts or livers 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). Protein concentration was determined by the Bradford or BCA method using BSA as a standard. Mitochondria (2 mg.ml-1) were lysed by addition of 200 µl of lysis buffer (100mM Tris-HCl, 100mM NaCl, 40mM MnCl2, 2mM dithiothreitol pH 7.5, 0.1% TritonX-100). The concentration of the RNase A (10 U/µl), RNase T1 (0.1 U/µl) or RNase If (0.01 U/µl) were optimized and added to each mitochondrial lysate or purified RNA to generate 15-55 nt size fragments. All incubations were carried out at 37˚C for up to 30 minutes and reactions were ended by addition of 700 µl Qiazol, followed by RNA isolation using the miRNeasy Mini Kit (Qiagen). We used mitochondria lysed and mock treated as controls for the footprinting assay.
The libraries were constructed using the Illumina TruSeq Small RNA Sample Prep Kit and deep sequencing of the mitochondrial small RNAs was performed by Australian Genomic Research Facility (Melbourne, Australia) on an Illumina GAII (Illumina, CA) according to the manufacturer’s instructions with one modification, sample isolation from the PAGE gel after adaptor ligation was performed with a modified set of size markers to facilitate capture of small RNAs between 15-55 nt.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
mitochondrial RNA
processed data files:
LRPPRC_KO.CPM.fwd.Cscore.wig
LRPPRC_KO.CPM.rev.Cscore.wig
LRPPRC_KO_A-T1.fwd.Rscore.wig
LRPPRC_KO_A-T1.rev.Rscore.wig
LRPPRC.KO_called.sig_footprints.bed
LRPPRC.WT_called.sig_footprints.bed
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| Data processing |
Technical replicates were pooled and sequenced reads were trimmed of adapter sequences with cutadapt v1.10, using default parameters, and aligned to the mouse genome (mm10) with Bowtie239 v2.2.9 with a seed length of 10 and reporting up to 20 alignments per read (-L10 -k 20).
Paired alignments with multiply mapping reads that aligned once to the mitochondrial genome and at least once to a NUMT region were rescued.
All properly paired alignments to the mitochondrial genome with an observed template length of 15 to 35 nt were retained and a subtractive alignment against nuclear tRNA and Illumina contaminant sequences was performed.
Strand-specific fragment BED files were created and 5′ coverage profiles normalised to sequenced library size were produced with bedtools40 v2.26.0.
Footprints were identified as previously described (Liu et al., Cell Reports, 2013) with some modifications reported here. The RNase accessibility of each base, i, in the mitochondrial genome was quantified according to its C score, defined as: Cscorei = log10( max[Ai+1,T1i+1,Ifi+1] +1 / Untreatedi+1 +1 ) where Ai+1, T1i+1, Ifi+1 and Untreatedi+1 represent the normalised 5′ coverage of the nucleotide immediately downstream of the inspected position in RNase A-, T1-, and If-treated and untreated samples, respectively. The footprint detection algorithm searches for a span of between 8 and 40 nucleotides with an average C score lower than the flanking left and right three nucleotides, the central footprinting region, and calculates its F score: Fscore = 10C/10L + 10C/10R where C, L and R are the average C score of the central, left flanking and right flanking regions, respectively. We initially searched for footprints in the control data and for each footprint we calculated its F score(control) and the F score of the equivalent region in the knockout sample, F score(knockout), and the log2-transformed fold change of F scores (knockout/control). We also searched for novel RBP interactions or sites of decreased RNase accessibility by searching for footprints in the experimental data initially, before calculating F scores and extracting the data from the equivalent region in the control sample. To estimate if the F score fold change of a footprint is significant, we built an empirical null model by shuffling the C score of both samples within the mitochondrial genome 1,000 times and for each locus, calculating an F score fold change from each pair of shuffled data sets. For the candidate footprint loci, the central footprint region was required to have a C score(knockout) greater than its C score(control), and the F score fold change was filtered to achieve an expected 5% false discovery rate (FDR) relative to a score obtained from random shuffling 1,000 times, as previously described (Chen et al., Bioinformatics, 2010). The propensity for secondary structure was quantified according to the ratio of double-stranded RNase cleavage over single-stranded, its R score: Rscorei = log2( Ai+1 +1 / T1i+1 +1 )
Genome_build: mm10
Supplementary_files_format_and_content: WIG files containing per-base Cscore and Rscore values, and BED files containing significant footprint sites
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| Submission date |
Jul 03, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Stefan J Siira |
| Organization name |
The Kids Research Institute Australia
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| Department |
Precision Health
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| Lab |
Mitochondrial Medicine and Biology
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| Street address |
15 Hospital Ave, Nedlands
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| City |
Perth |
| State/province |
Western Australia |
| ZIP/Postal code |
6009 |
| Country |
Australia |
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| Platform ID |
GPL9250 |
| Series (2) |
| GSE100729 |
LRPPRC-mediated folding of the mitochondrial transcriptome [RNase footprinting] |
| GSE100733 |
LRPPRC-mediated folding of the mitochondrial transcriptome |
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| Relations |
| BioSample |
SAMN07311950 |
| SRA |
SRX2978829 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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