RNA-seq experiment and data analysis

RNA from hANOs on Days 10, 17, and 24 was isolated by using Trizol reagent (Thermo Fisher Scientific). RNA quality was checked by Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA). The library preparation was carried out following the manufacturer’s protocol by using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEW ENGLAND BioLabs, Ipswich, MA). cDNA libraries were sequenced on an Illumina Novaseq platform as 150‐bp pair ended reads (illumina, San Diego, CA).

Kallisto program was used to quantify the abundances of transcripts from RNA-seq data⁵⁵, and the Kallisto index was built with reference transcriptome GRCh38. Reads per kilobase per million mapped fragments (RPKM) counts and differential expression of gene transcripts were estimated using DEseq2 program (Bioconductor, https://www.bioconductor.org). The difference of transcriptomic data were analyzed by two-sided Wald test methods and the multiple comparison correction were analyzed by Benjamini–Hochberg methods. Differentially expressed genes (DEGs) were selected based on the cut-off at a P value of less than 0.05 and the absolute value of Log2 fold change greater than or equal to 1.5. For intra-group analysis, i.e., different differentiation days of hANOs, DEGs were analyzed in samples from Day 10, Day 17, and Day24 at pairwise comparison. For inter-group analysis, i.e., hANOs treated with MMP inhibitor versus untreated hANOs, DEGs were analyzed between MMP inhibitor treated and untreated samples on Day 17 or Day 24. ClusterProfiler program⁵⁶ was used to perform enrichment analysis, including Gene Ontology (GO) term analysis, KEGG pathway analysis and GSEA analysis based on the DEGs from above intra-group or inter-group comparisons; the above functional analyses were analyzed by two-sided Over Representation Analysis (ORA) with Benjamini–Hochberg multiple comparison correction.