m6A modification segregates the stemness program from proliferation in ISCs [RNA-seq]

Purpose: One goal of this Bulk RNA-seq is to compare genome-wide transcriptome profiles in WT and Mettl3-KO mice intestinal stem cells at indicated time point.The other goal is to compare expression of stemness genes in WT, Mettl3-KO, Mettl3 KO and AKF-OE or AKFP-OE organoids. Methods: For mice, intestinal crypts were isolated and digested with Tryple, Lgr5-high or Lgr5-low cells were sorted by flow cytometry. For organoids, organoids were digested with Tryple and alive cells were sorted by flow cytometry. Total RNA was extracted with RNeasy Mini Kit (QIAGEN) according to the manufacturer's instructions. Bulk RNA-seq was performed using the Illumina Hiseq X. RNA-seq reads were aligned to the mouse genome available in Ensembl (release 95) with HISAT2. Conclusions: Mettl3 deletion results in reduced expression of most of the stem cell signature genes in Mettl3-KO ISCs, including Lgr5, Olfm4, Ascl2, and Smoc2, but increased gene expression related to cell death, p53, MAPK, YAP and regeneration pathway. AKF and AKFP-overexpression could rescue the expression level of some ISC signature genes compared with Mettl3 detion organoids. Overall design: mRNA profiles in Lgr5-high or Lgr5-low cells from WT and Mettl3-KO mice at indicated time; mRNA profiles in Intestinal epithelial cells from WT, Mettl3-KO, Mettl3-KO-AKF-OE, Mettl3-KO-AKFP-OE organoids.