GSM8996769: GFPF24 sorted GFP+ Foxl1+ telocytes; Mus musculus; RNA-Seq
Isolation of FOXL1+ cells using FACS was performed using Foxl1-cre;Rosa26- mTmG mice. Jejunums were dissected and washed thoroughly with Hank's balanced salt solution (HBSS) and were incubated in 5 mM EDTA in HBSS for 10 min at 4 °C. Intestinal villi were scraped off using a coverslip and the remaining tissue was cut into small pieces and incubated in 5 mM EDTA and HBSS on ice for 10 min while pipetting to completely remove the remaining epithelium. After vigorous washes, the remaining mesenchymal fraction was incubated with 6 mg/ml Dispase II/0.05% trypsin solution (Sigma-Aldrich, 04942078001) supplemented with 1 U/ml DNaseI (Invitrogen) at 37 °C, until the solution became cloudy and the mesenchyme was dissociated (15 min). Supernatant was filtered through a 70-μm strainer, centrifuged and resuspended with anti CD45–APC and anti EPCAM–APC antibodies (1:300, Biosciences, and 1:100, BioLegend, respectively) in 10%FBS/5 mM EDTA/HBSS for 45 min at room temperature to exclude immune and epithelial cells from sorting. A single-cell suspension was obtained by filtering the supernatant through a 40-μm strainer and washing with 5 mM EDTA in HBSS, before cell sorting. Cells were loaded onto a Chromium single-cell 3′ v2 chip (10x Genomics) or Chromium GEM-X Single Cell Gene Expression v4 (3') (10x Genomics) and libraries were prepared according to the manufacturer's protocol.