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| Status |
Public on May 04, 2022 |
| Title |
MicroRNA-486 is an epigenetic modulator of Duchenne muscular dystrophy pathologies [eCLIP-seq] |
| Organism |
Mus musculus |
| Experiment type |
Other
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| Summary |
Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle disorder resulting in muscle weakness and cardiomyopathy. MicroRNAs have shown to play a significant role in muscle development, metabolism, and disease pathologies. We demonstrated that miR-486 expression is reduced in DMD muscles and its expression levels correlate with dystrophic disease severity. miR-486 knockout mice developed disrupted myofiber architecture, decreased myofiber size, decreased locomotor activity, increased fibrosis, and metabolic defects that were exacerbated on the dystrophic mdx5cv background. We integrated RNA-sequencing and chimeric eCLIP-sequencing data to identify direct in vivo targets of miR-486 and associated dysregulated gene signatures in skeletal muscle. Together, our studies identify miR-486 as a driver of muscle remodeling in DMD, a useful biomarker for dystrophic disease progression, and highlight chimeric eCLIP-sequencing as a useful tool to identify direct in vivo microRNA target transcripts.
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| Overall design |
Whole TA muscles were isolated from male 6-month-old wild type and miR-486 KO mice (n = 3 total samples per genotype; performed in duplicate) and snap frozen in liquid nitrogen. The samples were processed for chimeric eCLIP-sequencing by Eclipse Bioinnovations Inc. (San Diego, CA). Approximately 80 mg of mouse muscles were homogenized by cryogenic pulverization. Samples were then were UV (254 nm) cross-linked twice at 400 mJ/cm2 using Stratalinker 2400 (Stratagene; San Diego, CA), on a bed of ice. Following cross-linking, the samples were sonicated (QSonica Q800R2; QSonica LLC, Newtown, CT) to shear the genomic DNA into smaller fragments. Ago2 immunoprecipitation using an Ago2 antibody (EclipseBioinnovations Inc.) was pre-coupled to anti-mouse Dynabeads (M-280 Sheep Anti-Mouse IgG Dynabeads, ThermoFisher Scientific 11201D), added to the homogenized lysate, and incubated overnight at 40C with gentile rocking. After immunoprecipitation, 2% of the sample was taken as the paired input sample. For chimeric eCLIP experiments, a standardized eCLIP protocol2 was modified to enable chimeric ligation of the miRNA and mRNA (Van Nostrand, E & Yeo, G, personal communication). miRNA-specific chimeric-eCLIP was performed by amplifying cDNA using a mouse miR-486a primer and a sequencing adapter-specific primer, following amplification with indexed primers. Chimeric eCLIP-sequencing Library Preparation: Whole TA muscles were isolated from male 6-month-old wild type and miR-486 KO mice (n = 5 total samples per genotype; performed in duplicate) and snap frozen in liquid nitrogen. The samples were processed for chimeric eCLIP-sequencing by Eclipse Bioinnovations Inc. (San Diego, CA). Approximately 80 mg of mouse muscles were homogenized by cryogenic pulverization. Samples were then UV (254 nm) cross-linked twice at 400 mJ/cm2 using Stratalinker 2400 (Stratagene; San Diego, CA), on a bed of ice. Following cross-linking, the samples were sonicated (QSonica Q800R2; QSonica LLC, Newtown, CT) to shear the genomic DNA into smaller fragments. Ago2 immunoprecipitation using an Ago2 antibody (EclipseBioinnovations Inc.) was pre-coupled to anti-mouse Dynabeads (M-280 Sheep Anti-Mouse IgG Dynabeads, ThermoFisher Scientific 11201D), added to the homogenized lysate, and incubated overnight at 40C with gentile rocking. After immunoprecipitation, 2% of the sample was taken as the paired input sample. For chimeric eCLIP experiments, a standardized eCLIP protocol was modified to enable chimeric ligation of the miRNA and mRNA (Van Nostrand, E & Yeo, G, personal communication). miRNA-specific chimeric-eCLIP was performed by amplifying cDNA using a mouse miR-486a primer and a sequencing adapter-specific primer, following amplification with indexed primers.
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| Contributor(s) |
Rylie H, Matthew A |
| Citation(s) |
35512829 |
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https://doi.org/10.1101/2021.06.14.448387
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| Submission date |
May 04, 2021 |
| Last update date |
Jun 01, 2022 |
| Contact name |
Matthew S Alexander |
| E-mail(s) |
malex3@uab.edu
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| Organization name |
University of Alabama at Birmingham
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| Department |
Pediatrics
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| Lab |
Matthew Alexander
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| Street address |
1900 University Blvd. THT 929 Box# 96
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| City |
Birmingham |
| State/province |
AL |
| ZIP/Postal code |
35294 |
| Country |
USA |
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| Platforms (1) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
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| Samples (12)
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| This SubSeries is part of SuperSeries: |
| GSE178778 |
miR-486 is an epigenetic modulator of Duchenne muscular dystrophy pathologies |
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| Relations |
| BioProject |
PRJNA727188 |
| SRA |
SRP318395 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE173821_0096_IP_1_S3_01.basedon.peaks.l2inputnormnew.bed.compressed.bed.gz |
1.0 Mb |
(ftp)(http) |
BED |
| GSE173821_0096_IP_3_S5_01.basedon.peaks.l2inputnormnew.bed.compressed.bed.gz |
1.3 Mb |
(ftp)(http) |
BED |
| GSE173821_0096_KO_vs_WT_01.basedon.peaks.l2inputnormnew.bed.compressed.bed.gz |
1.3 Mb |
(ftp)(http) |
BED |
| GSE173821_0096_WT_vs_KO_01.basedon.peaks.l2inputnormnew.bed.compressed.bed.gz |
1.1 Mb |
(ftp)(http) |
BED |
| GSE173821_152_AlKo_01.basedon.peaks.l2inputnormnew.bed.compressed.bed.gz |
2.9 Mb |
(ftp)(http) |
BED |
| GSE173821_152_AlKo_vs_WtAl_01.basedon.peaks.l2inputnormnew.bed.compressed.bed.gz |
2.9 Mb |
(ftp)(http) |
BED |
| GSE173821_152_WtAl_01.basedon.peaks.l2inputnormnew.bed.compressed.bed.gz |
2.8 Mb |
(ftp)(http) |
BED |
| GSE173821_152_WtAl_vs_AlKo_01.basedon.peaks.l2inputnormnew.bed.compressed.bed.gz |
2.9 Mb |
(ftp)(http) |
BED |
| GSE173821_RAW.tar |
395.0 Mb |
(http)(custom) |
TAR (of BW) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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