GSE132565
Whole transcriptomes analyses of intestinal epithelial cells (IECs) isolated from Mst4F/F Villincre (Mst4 KO) mice and wildtype (WT) mice
Purpose: To explore the molecular mechanisms of MST4 regulating intestinal stem cell homeostasis, RNA sequencing was performed to analyze the geome-wide change of MST4 KO IECs compared to those of WT mice. Methods: Total mRNA was extracted from IECs which were isolated from small intestines of Mst4 KO mice and WT mice (8 weeks) in triplicate respectively. Then RNA quality was assessed using an Agilent Bioanalyzer 2100 and the sample reads were sequenced using Illumina Hiseq 4000 platform. As a reasult, we got the transcript data using Hisat2 followed by Stringtie. qRT–PCR validation was performed using TaqMan and SYBR Green assays. Results: We mapped about 60 million sequence reads per sample to the mouse genome and identified about 50,000 transcripts in IECs of mice. Apprioximately 2,000 transcripts showed different expression between Mst4 KO and WT IECs, with a fold change =2 and p value <0.05. Gene set enrichment analysis (GSEA) showed a significantly negative enrichment of Wnt/b-catenin target genes and altered expression of 15 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Conclusion: Our study present the datiled transcripts analysis of  IECs isolated from small intestines, with biologic replicates. Based on RNA-seq transcriptome characterization , we conclude a molecular mechanism of MST4 regulating intestinal stem cell homeostasis and provide a  framework to understand the crosstalk of MST4 and Wnt/b-catenin signaling. Overall design: Total mRNA was extracted from IECs of Mst4 KO and WT mice (about 8 weeks) in triplicate. The quality was assessed using Agilent 2100, and the profiles were sequenced on the Illumina Hiseq X10.