GSM8940944: Mouse Gut EEC 28; Mus musculus; RNA-Seq
EEC protocol: Samples included Neurod1-Cre and Neurog3-Cre mice that were crossed with Ai9 reporters, Chga-GFP, Tph1-CFP, Ffar3-mRFP, and wild type mice. Intestines (stomach, duodenum, jejunum, ileum, and colon) were digested to single cell suspensions following the protocol described in Roberts et al (Diabetes, 2019). They were then FACSed sorted to the cells that emit the fluorophore or wild type mice were sorted with antibodiesto Epcam+/Cldn- cells. For two libraries (K_10X_mGUT035 and K_10X_mGUT036), cell hashing was employed to multiplex multiple samples following the protocol at https://www.protocols.io/view/cite-seq-5qpvo4rxg4o1/v1  Nodose protocol: Wild type mice were used. Nodose ganglia were dissected and as much nerve as possible we trimmed off. 10-11 ganglia were pooled and dissociated with 500 ul of dissociation solution, (5 mg/ml Collegenase II,1 mg/ml Dispace II, in Hank's buffered salt solution) and stored on ice. After dissections were complete, ganglia in dissociation solution were placed in a 37 C water bath for 1 hour with gentle mixing and pipetting every 10 minutes. Triteration was performed with a 1 ml pipette. Undissociated tissue was visualized by microscope to check for the presence of neurons. Dissociation continued until all neurons were visibly dissociated. Then, 500 ul of nodose medium (10% heat inactivated fetal bovine serum in DMEM) was added to the dissociated tissue, mixed 5 times with a 1 ml pippette and spun at 420 RCF for 5 minutes at 4 C. Supernatatants were discarded and pellets were resuspended in nodose medium. Resuspended cells were layered on top of 1ml of BSA gradient in DPBS on ice. Tubes with BSA and cell suspections were spun at 200 RCF for 5 minutes at 4 C. Supernatants were discarded; pellets were resuspended in 500 ul DPBS with 0.4% BSA and filtered through a 70 um cell strainer. Cells were counted and diluted following the 10X Genomics protocol Single cell suspensions were loaded on to a 10x Chromium Controller as quickly as possible. Approximately 20,000 cells were loaded per reaction. LIbraries were generated with V2 or V3 chemistry following 10x Genomics protocols.