Transcriptomic profiling of small intestinal crypts upon MEX3A deletion

The effort to better understand intestinal stem cell (ISC) identity and regulation remains a challenge. We have been studying the RNA-binding protein MEX3A as a putative ISC marker. In that context, we have generated the first Mex3a knockout (KO) mouse model and show MEX3A is crucial for maintenance of the Lgr5+ ISC pool. As part of a phenotypic characterization pipeline, we have performed transcriptomic profiling (RNA-sequencing) of isolated Mex3a KO small intestinal crypts and compared it against small intestinal crypts isolated from age-matched wild-type controls. Given the effect of Mex3a deletion for crypt cell populations and overall crypt development, Mex3a KO animals were sacrificed before the peak of phenotypic onset in order to preserve the crypt cellular content to its maximum extent. For each biological replicate (n = 3 for each genotype at P16), small intestinal crypts were isolated by incubation of intestinal fragments with crypt chelating buffer, followed by mechanical dissociation. Freshly isolated crypts were used for total RNA extraction with Tri reagent (Sigma) and subsequent RNA-sequencing analysis.