Single-cell RNA Sequencing of 3D MSCs

In this study, 3D MSCs were obtained, and single-cell suspensions were collected. Based upon the Singleron Matrix single-cell processing system, single-cell suspensions of 3D MSCs are loaded onto a microwell chip. Subsequently, the captured mRNA is reverse-transcribed and the cDNA was amplified, fragmented and ligated to sequencing adapters. Constructed by the GEXSCOPE Single Cell RNA Library Kit (Singleron), the individual libraries of 3D MSCs were mixed and sequenced by the Illumina platform (novaseq 6000). With the obtained sequencing data, bioinformatics analysis techniques such as t-SNE, PCA, or clustering methods were applied to analyze the differences between individual cells using Seurat package (25 (link)), which allowed for a more in-depth exploration of the changes in single-cell biology features, development, and functional variations of 3D MSCs. COSine similarity-based marker Gene identification (COSG) package was utilized to calculate the markers of each clusters (26 (link)) and GO, KEGG and Reactome enrichment analyses were done to better understand the heterogeneity of MSCs.