Stem loop reverse transcription primers

Total RNA from cell cultures was extracted using the semi-automated Maxwell RSC simplyRNA tissue kit (Promega) or from frozen mouse crural muscles (soleus and gastrocnemius) with TRIzol Reagent (Thermo Fisher). Prior to RNA isolation, EVs underwent pre-treatment with proteinase/RNase to eliminate co-precipitated free RNA. Then, total RNA was extracted using Direct-Zol RNA Miniprep kit (Zymo Research) following the manufacturer instructions. 1 μg of total RNA was reverse transcribed with random primers and Moloney murine leukemia virus reverse transcriptase (Thermo Fisher Scientific). For miRNAs, 5 ng of total RNA was retro-transcribed with stem-loop reverse transcription primers (Thermo Fisher Scientific) and MultiScribe reverse transcriptase enzyme (Thermo Fisher Scientific). qPCRs were performed on a ViiA 7 Real-Time PCR System (Thermo Fisher Scientific) using PrimeTime qPCR gene expression assays (IDT) for the protein-coding transcripts and TaqMan® MicroRNA Assays (Thermo Fisher Scientific) for the miRNAs (Supplemental Table 1). β-actin and GAPDH were used as housekeeping genes for mRNA analysis in mouse and human samples respectively. RNU6-1 (codifying for U6 snRNA) was used as reference gene to normalize miRNA readouts for mouse samples, and miR-103a for human samples.