Preparation and analysis of RNA-seq libraries

Total RNA was extracted from IMRCs and UCMSCs using Trizol (Invitrogen, Waltham, MA, USA; 15596018). Subsequently, RNA-seq libraries were prepared with the NEBNext^® Ultra^TM RNA Library Prep Kit for Illumina^® and subjected to paired-end sequencing with 150 bp reads on an Illumina HiSeq X-Ten sequencer. After filtering the sequencing data, we utilized STAR to map them to the hg38 reference genome. Gene expression levels were estimated by counting the reads mapped to genomic or exon regions, and FPKM (Fragments per Kilobase per Million Mapped Fragments) was used. DESeq2 was employed for differential gene expression analysis, with criteria set as |log2-fold change | ≥1 and P value < 0.05. Principal Component Analysis (PCA) was performed using the DESeq2 package in R. Gene Ontology analysis for DEGs was conducted using DAVID (version 6.8). Heatmap analysis was carried out using the heatmap.2 functions in R.