design: Rna-Seq expression profiling of Defa6+ cells possessing either wild-type Tcf4 or heterozygous and homozygous loss of Tcf4 protein isolated from mouse small intestinal epithelium
library_construction_protocol: Adult littermates of the Rosa26tdTomato Defa6-iCre mouse strain carrying the Tcf7l2-wt or Tcf4-floxed alleles were sacrificed by cervical dislocation. In these mice, cells expressing Defa6 producing constitutively active Cre recombinase are marked by endogenous expression of the red fluorescent protein tdTomato. The distal part of the small intestinal epithelium was isolated, and a suspension of single cells was prepared by dispase cleavage. Cells were stained with the following antibodies: Hoechst33358, anti-CD45-PacificBlue, anti-CD31-PacificBlue, anti-EpCAM-FITC. Using fluorescence-activated cell sorting (FACS), we isolated Hoechst-/CD45-/CD31-/EpCAM+/tdTomato+ cells, i.e. Defa6+ cells with either wild-type Tcf4 or heterozygous and homozygous loss of Tcf4 protein. Total RNA was isolated using RNeasy Micro Kit (Qiagen) as described in the manufacturer's protocol. Libraries were prepared with a SMARTer® Stranded Total RNA-Seq – Pico Input Mammalian library preparation kit v2 (Takara).