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).

本研究从血浆样本中提取总RNA，所用试剂为Qiagen血清/血浆RNA提取试剂盒（Qiagen, 希尔德，德国，美国）。采用安捷伦2100生物分析仪（Agilent Technologies, 加利福尼亚州，美国）对RNA的质量与完整性进行评估。采用Qiagen QIAseq miRNA文库制备试剂盒（Qiagen, 德国，美国）构建下一代测序（NGS）文库，具体流程为依次在miRNA的3'端与5'端连接接头，随后基于唯一分子标识符（UMI）进行cDNA合成、cDNA纯化，并使用通用引物与索引引物完成扩增。采用安捷伦2100生物分析仪搭配高灵敏度DNA试剂盒（Agilent Technologies, 加利福尼亚州，美国）对文库质量进行检测。测序工作在Illumina NextSeq 6000测序平台（Illumina公司, 加利福尼亚州，美国）上完成。测序完成后，使用Fastx工具包（v0.0.13）过滤掉低质量读数与污染读数。使用Bowtie将过滤后的清洁读数比对至人类参考基因组GRCh38。基于miRBase数据库第21版鉴定已知miRNA，同时使用miRCat软件预测新miRNA。采用每百万转录本（TPM）值对基因表达水平进行定量。使用edgeR软件鉴定差异表达miRNA，筛选阈值设定为错误发现率（FDR）<0.05且绝对log2折叠变化（|log2(FC)|）>1。使用miRanda软件（http://www.microrna.org/）预测差异表达miRNA的靶基因。采用基因本体（GO）与京都基因与基因组百科全书（KEGG）通路富集分析对靶基因进行功能注释。文库构建与测序工作均在上海生物科技有限公司（中国上海）完成。