Intestinal endocrine subtype specification analyzed by Single Cell RNA-Seq in mouse enteroids/mini-guts.

The goal of this experiment is to decipher the mechanism of intestinal endocrine / enteroendocrine subtype specification, that is far from being fully understood. To this end, we used single cell genomics in mouse mini-guts. Enteroendocrine cells are derived from endocrine progenitors expressing the transcription factor Ngn3. We took advantage of the Ngn3+/eYFP mouse model -where endocrine progenitors and their descendants can be isolated and sorted by FACS on the basis of the eYFP fluorescence- and established enteroids or mini-guts from the small intestine. Enteroids were dissociated and eYFP+ single cells were directly sorted in 96 wells of the Precise WTA Single Cell Encoding Plate (BD™ Precise WTA Single Cell Kit, BD Genomics). cDNA and libraries were prepared following the BD protocol. Sequencing (paired-end, 2x100b) was performed in a HiSeq 4000 (Illumina). The bioinformatic analysis allowed to identify 8 different groups of enteroendocrine cells with specific signatures. Cells from Ngn3eYFP mice enteroids were sorted and captured. Their transcriptomes were analyzed by single-cell RNASeq.