Properties and functions of transcriptionally distinct enteric neurons
Animals from the indicated genotypes were generally sacrificed after 7-14 days of reporter gene expression. Animals were first perfused with 1x PBS and then intestine was rapidly dissected and stored in ice-cold dissection media (DMEM:F12 media supplemented with penicillin-streptomycin). Segments from each region of the intestine, namely duodenum, jejunum, ileum, proximal and distal colon, were subdissected and rinsed to remove any food residue or fecal matter. Tissue segments were then cut up into shorter segments and pinned to a sylgard coated glass petri dish. An incision was made along the length of the intestine segment and insect pins were used to anchor the tissue such that the villi were facing up. Sharp forceps were used to separate the mucosal layer from the enteric plexuses. Isolated plexuses were stored in dissection media on ice for the remainder of the dissection period. Dissection periods were timed and were conducted for no longer than 2 hours to maximize tissue integrity. The remaining layers of the intestine were kept in a microfuge tube con-taining ice-cold DMEM:F12. Isolated plexuses were dissociated in 1mg/ml Liberase TH, 20 unit/mL Dispase, 0.1mg/ml DNAseI in DMEM:F12 + 1% pen/strep for 30 mins at 37°C. Digestion was quenched in media containing 20mg/mL BSA and 5mM EDTA in DMEM:F12, 1% pen/strep. Cell preparations were strictly maintained on ice/4°C for the remainder of the protocol. Digested tissue was gently triturated with fire-polished glass pipettes (opening diameter of approximately 100μm). Tissue was then passed through a 70-μm filter to remove cell doublets and debris. The neurons were pelleted and washed 4 times in 20 mg/ml BSA in DMEM:F12 + 1% penicillin-streptomycin fol-lowed by 2x washes with DMEM:F12 + 1% pen/strep all at 4°C. After washing, Tissue were resus-pended in 45μl of DMEM:F12 + 1% Pen/strep, DyeCycle Ruby and Cytox Blue. Cells were sorted on a Sony MA-900 and gated on H2bmTagXFP expression, DyeCycle Ruby and Cytox Blue signal to ensure intact and viable whole cells were collected. Sympathetic ganglia were processed in a similar fash-ion with the following modifications. Dissected sympathetic ganglia were first collected in DMEM:F12 supplemented with 1% pen/strep and 12.5 mM D-glucose. Dissociations were per-formed in 40 units papain, 4 mg/ml collagenase, 10 mg/ml BSA, 1 mg/ml hyaluronidase, 0.6 mg/ml DNase in DMEM:F12 + 1% pen/strep + 12.5 mM glucose for 30 min at 37°C. Digestion was quenched using 20 mg/ml ovomucoid (trypsin inhibitor), 20 mg/ml BSA in DMEM:F12 + 1% pen/strep + 12.5 mM glucose. Dissociated ganglia tissue were washed in a similar fashion to enter-ic plexus peels and resulting single-cell suspensions resuspended in an appropriate volume of DMEM:F12 + 1% pen/strep + 12.5 mM glucose. In all conditions, collected intact whole single cells were directly encapsulated for cDNA synthesis using the 10x Genomics platform.  Enteric ganglia from mouse intestine were dissected, dissociated and isolated by FACS to obtain whole cell single cell RNA sequencing analysis.  Everything related to nucleic acid extraction was done in accordance with the 10x genomics recommendations. Everything related to nucleic acid extraction was done in accordance with the 10x genomics recommendations.