title: GSM8656650: GFP positive EECs; Mus musculus; RNA-Seq
library_construction_protocol: intestines were dissected out and bisected longitudinally to expose the luminal epithelia and submerged in cold dissociation cocktail (2.25 mg/mL DNAse (Sigma Cat. #: DN25), 10 mg/mL protease (Sigma Cat. #: P5380), and 9 µM Y-27632 (Selleck Chem. Cat. #: S1049) in PBS). Samples were incubated in dissociation cocktail on ice for 30 minutes with trituration every 5 minutes until achieving >90% single cells in the suspension. Cells were then rinsed 3x in cold PBS, passed through a 40 µm cell strainer, pelleted and resuspended in cold DMEM+10% FBS with 2.25% DNAse and 9 µM Y-27632 to preserve viability during cell sorting. Final cell counts were ~2.75e6 cells from E15.5 embryos, ~7.5e6 cells from E16.5 embryos and ~7.5e6 cells from E17.5 embryos, all of which were stained with Sytox and Annexin V (to label dead cells) and pooled prior to sorting. FACS isolation of GFP-POS EECs was performed on a Sony SH800 cell sorter with gating based on  GFP-NEG controls (one Cre-NEG ROSAmTmG littermate from each age), excluding SytoxPOS and Annexin VPOS cells. Sorted ~65,000 GFP-POS cells and viability was determined to be ~89% by LUNA-FL Cell Counter before library preparation. 10,000 single cells were processed through the Chromium Next GEM Single Cell 3' Reagent Kit v3.1 (Dual Index) with Feature Barcode technology for Cell Surface Protein (10X Genomics, Pleasanton, CA) as per the manufacturer's instructions. Single-cell cDNA libraries were sequences on the Illumina NextSeq 2000 platform with a NextSeq P3 flow cell.