Exoquick exosome rna column purification kit

RNA expression was detected through qRT‐PCR. To isolate total RNA, TRIzol™ Reagent (Invitrogen) was adopted, while ExoQuick® Exosome RNA Column Purification Kit (System Biosciences, Palo Alto, CA, USA) was adopted for exosomal RNA extraction. DNase I, Amplification Grade (Invitrogen) was applied to digest DNA contaminants. Nanodrop™ 2000 spectrophotometer (Thermo Scientific) was used to detect the concentration and quality of RNA samples. RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) (for mRNA and lncRNA) and TaqMan MicroRNA Assays (Applied Biosystems, Waltham, MA) (for miRNA) was applied for synthesizing complementary DNAs (cDNAs) from 1 μg of RNA samples. qRT‐PCR was performed on CFX96 Touch Real‐Time PCR Detection System (Bio‐Rad, Hercules, CA, USA) using SYBR™ Green PCR Master Mix (Applied Biosystems). All data were normalized to the internal control GAPDH or U6, and 2^–ΔΔCt method was used for relative expression quantification. The sequences of qRT‐PCR primers (RiboBio) are presented in Supporting information Table S2.

RNA from EVs was extracted using ExoQuick Exosome RNA Column Purification Kit (EQ808A-1, System Biosciences, CA, USA) and miRNA library synthesis was performed using the QIAseq miRNA Library Kit (QIAGEN, Germany) following manufacturer instructions (QIAseq miRNA Library Kit Handbook ver. 03/2020). Prior to sequencing, the libraries underwent electrophoretic control on DNA1000 capillary electrophoresis cartridges of Bioanalyzer 2100 (Agilent Technologies, Italia), and quantification by Qubit fluorimetric DNA High Sensitivity Assay (Thermo Fisher Scientific, USA). The libraries have been sequenced in 76 bp Single End run on a NextSeq 500 platform (Illumina, USA, RRID:SCR_017958) with automated trimming and demultiplexing made online to the sequencing with the Illumina Cloud BaseSpace Sequencing Hub.

RNA isolation was performed on the muscle myobundles and EVs prior to submitting samples for miRNAseq at the Duke University Center for Genomic and Computational Biology. RNA was extracted from myobundles using the Aurum^™ Total RNA Mini Kit (Bio-Rad; Hercules, CA, United States) in accordance with manufacturers specification. Extraction of RNA from EVs was performed using ExoQuick^® Exosome RNA Column Purification Kit (System Bioscience; Palo Alto, CA, United States) in accordance with the manufacturer’s specifications. RNA was subsequently dissolved in 20 μL RNAse-free water and underwent quantification via the Qubit^™ RNA HS Assay Kit (Thermo Fisher Scientific; Waltham, MA, United States) according to the manufacturer’s specifications.
Library preparation of myobundle and EV RNA samples was completed using the NextSeq 500 Mid-Output Library Kit (Illumina Inc.; San Diego, CA, United States). Following library preparation, sequencing was completed on the Illumina NextSeq500 (Illumina; San Diego, CA, United States) yielding 75bp paired-end reads at a depth of 16 M.