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

Purpose: A method for identifying genome-wide DNA binding sites for Fosb. Methods: Alive cells were sorted from retro-Fosb-OE(over-expression) organoids. The samples were incubated with anti-Fosb antibody (Abcam, ab184938). Purified DNA was subjected to Tru-seq library construction using NEBNext Ultra II DNA Library Prep Kit and sequenced as paired-end with Illumina Novaseq 6000. HISAT2 was used to align the sequences to the mouse genome and generate bam files. bamCoverage (CPM normalized and extended reads) was used to generate bigwig files from bam files. MACS2 was used for peak calling and to generate bed files from aligned reads. HOMER annotatePeaks.pl was used to annotate the peaks. Conclusions: Target genes of Fosb through ChIP assay were consistent with predicted target genes. Thus, we concluded that Fosb, which is a key TF could regulate most of ISC signature genes to maintain Lgr5+ ISCs. Overall design: Examination of Fosb binding sites and target genes in Intestinal epithelial cells.