Comparing BMMSCs treated with PBS (Con group) or EM-eNMs (EM-eNMs group)

RNA sequencing. Total RNAs from aged BMMSCs (P10-12), with or without em-eNMs treatment, were extracted by Trizol reagent (Thermo Fisher Scientific, Cat. No. 15596026). Sequencing libraries were generated using the NEBNext(R) Ultra(TM) RNA Library Prep Kit for Illumina(R) (NEB, USA) following the manufacturer's recommendations. Samples were then sequenced on an Illumina HiSeq 3000 platform. Quality control of the RNA-seq data was performed using FastQC (v0.11.5) and the FASTX toolkit (0.0.13). The data were mapped to the human reference genome (NCBI build 37, Jul 2007, mm9) using HISAT2 (v.2.0.4). Three samples per group were used. Differentially expressed genes and transcripts were identified using Ballgown software (v.3.4.0). Genes were considered significantly differentially expressed if they showed a >=1.5-fold change and a P value < 0.05.RNA测序。本研究提取经或未经em-eNMs处理的传代10-12代衰老骨髓间充质干细胞（Bone Marrow Mesenchymal Stem Cells, BMMSCs）的总RNA，提取采用Trizol试剂（赛默飞世尔科技，货号15596026）。参照厂商推荐操作流程，使用NEBNext® Ultra™ Illumina® RNA文库制备试剂盒（美国纽英伦生物技术公司，NEB）构建测序文库。随后在Illumina HiSeq 3000测序平台完成样本测序。采用FastQC（v0.11.5）与FASTX工具包（v0.0.13）对RNA测序数据进行质量控制。使用HISAT2（v2.0.4）将测序数据比对至人类参考基因组（NCBI构建37，2007年7月，mm9）。每组设置3个生物学重复样本。使用Ballgown软件（v3.4.0）鉴定差异表达基因与转录本，当基因表达变化倍数≥1.5且P值<0.05时，判定为显著差异表达基因。