Table 1.

Methods Used to Isolate Cells or Nuclei for RNA-Seq

Genotype	Whole Cells, or Nuclei?	Description
Wnt1-Cre;R26R-EGFP	Cells	The EGFP signal in Wnt1-Cre;R26R-EGFP animals was too weak to flow sort effectively given the high degree of background in the 488 channel.
Wnt1-Cre;R26R-tdTomato	Cells	The localization of tdTomato to neurites in our Wnt1-Cre;R26R-Tdtomato line was problematic, since we desired clean separation of single cells. Sorting myenteric plexus from this mouse line often resulted in preps with neurites attached to tdTomato- cells. We tried dissociating with different proteases (cold active protease, dispase, and collagenase), different incubation times (15 min, 30 min), multiple methods of trituration (pipette-based, needle-based), and different bowel layers, with little improvement in outcome
Wnt1-Cre;ROSANT-NG	Nuclei	Wnt1-Cre;RosaNT-NG mice had tdTomato in their nuclei at baseline; with CRE-induced recombination, they accumulated EGFP in their nuclei instead of tdTomato. Unfortunately, these mice lost fluorescent signal during the Dounce homogenization procedure. We hypothesize that membrane damage associated with homogenization led to diffusion of GFP and loss of signal.
Wnt1-Cre;Rosa26 LSL H2B mCherry	Nuclei	Successful and used to generate data in Figure 1, Figure 2, Figure 3, Figure 4.
Wild-type	Nuclei	We attempted to use directly conjugated NeuN and PHOX2B antibodies to isolate mouse ENS nuclei with flow sorting, since some neuronal nuclei in mouse stain with this NeuN antibody by immunohistochemistry. We were unsuccessful.

EGFP, enhanced green fluorescent protein; ENS, enteric nervous system; GFP, green fluorescent protein; RNA-seq, RNA sequencing.