EV-NGS: miRNA contents in EVs
Total RNA was extracted from plasma using the Qiagen Serum/Plasma Kit (Qiagen, Hilden, DE, USA). RNA quality and integrity were assessed with an Agilent 2100 BioAnalyzer (Agilent Technologies, CA, USA). Next-generation sequencing (NGS) libraries were prepared using the QIAseq miRNA Library Kit (Qiagen, DE, USA). The protocol involved sequential ligation of adapters to the 3' and 5' ends of miRNAs, followed by unique molecular identifier (UMI)-based cDNA synthesis, cleanup of cDNA, and amplification using universal and indexing primers. Library quality was measured with a 2100 BioAnalyzer using the High Sensitivity DNA Kit (Agilent Technologies, CA, USA). Sequencing was performed on an Illumina NextSeq 6000 platform (Illumina, Inc., CA, USA). Post-sequencing, low-quality and contaminant reads were removed using Fastx toolkit (v0.0.13). Clean reads were mapped to the human reference genome (GRCh38) using Bowtie. Known miRNAs were identified using miRBase release 21, while novel miRNAs were detected with miRCat. Gene expression levels were quantified using transcripts per million (TPM) values. Differentially expressed miRNAs were identified using edgeR with a false discovery rate (FDR) threshold of <0.05 and absolute log2 fold change (|log2(FC)|) >1. Target genes for differentially expressed miRNAs were predicted using miRanda (http://www.microrna.org/). Functional annotations of target genes were performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Library preparation and sequencing were conducted at Shanghai Biotechnology Corporation (Shanghai, China).