miRNAs (10 ng) were reverse transcribed using the Ion Total RNA‐seq kit v2 (4475936, Thermo Fisher Scientific) following the manufacturer's protocol for small RNA libraries. The cDNA libraries were amplified and barcoded using Ion Total RNA‐seq kit v2 and Ion Xpress RNA‐seq Barcode Adapters 1‐16 Kit (Thermo Fisher Scientific). Amplicons were quantified using Agilent High Sensitivity DNA kit before samples pooling in sets of 15. Emulsion PCR and enrichment were performed on the Ion OT2 system instrument using the Ion PI Hi‐Q OT2 200 kit (A26434, Thermo Fisher Scientific). Samples were loaded on an Ion PI v3 Chip and sequenced on the Ion Proton System using Ion PI Hi‐Q sequencing 200 kit chemistry (200 bp read length; A26433, Thermo Fisher Scientific). Sequencing reads were trimmed with Prinseq 33 (v0.20.4) (‐‐trim‐right 20) and filtered by average quality score (‐‐trim‐qual 20). Reads with a size less than 15 bp were removed and reads with a size greater than 100 bp were trimmed with Cutadapt (v1.16). 34 Mapping to the human genome assembly Ensembl GRCh37.87 (3111 transcripts) was performed with STAR 2.5.3a. 27 Normalized counts (median ratio normalization) and differential expression analysis were performed with DESeq2 1.16.1, 30 considering pairwise comparisons with all developmental stages and comparing DMD vs. healthy cells within developmental stages. Transcripts with |log2FoldChange| ≥ 0.4 (equivalent of DMD/healthy ratio ≤0.76 or ≥1.32) and P value ≤ 0.05 were considered differentially expressed. The use of P value instead of adjusted P value was justified by biological meaning 35 (i.e. well‐known regulated/dysregulated miRNAs had a P value ≤ 0.05 but not an adjusted P value ≤ 0.05). miRNA‐seq data have been deposited in the ArrayExpress database 32 at EMBL‐EBI under accession number E‐MTAB‐8293 (https://www.ebi.ac.uk/arrayexpress/experiments/E‐MTAB‐8293).

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