Expression analysis of adult zebrafish enteric nervous system

The enteric nervous system (ENS) encompasses the intrinsic neuroglia networks of the gastrointestinal (GI) tract that are essential for digestive function and gut homeostasis. To investigate the ENS of zebrafish, we carried out bulk RNA sequencing on nuclei purified by FACS (fluorescent-activated cell sorting) representing both the Cherry+ (ENS) and Cherry- (non-ENS) muscularis externa cell populations of Tg(sox10:Cre;Cherry) zebrafish gut. Overall design: The smooth muscle layers and associated myenteric plexus were separated from the epithelial layers of the adult gut and subjected to Dounce homogenization. After filtering the homogenate to remove large debris, samples were centrifuged at 1000 x G for 10 min at 4°C to obtain a pellet containing muscularis externa nuclei. For flow cytometric analysis, doublet discrimination gating was applied to exclude aggregated nuclei, and intact nuclei were determined by subsequent gating on the area and height of DAPI intensity. Both Cherry+ (ENS) and Cherry- (non-ENS) nuclear populations were collected directly into 1.5mL tubes containing Trizol LS reagent. For each replicate, sorted cells from an average of 30 adult guts were pooled, containing approximately 30,000 Cherry+ or Cherry- nuclear populations.