GSM8409616: large intestine, 9w, scRNA-seq, lane2; Mus musculus; RNA-Seq
Intestines were collected, cut into 1cm pieces, and then washed with EDTA-free cold PBS. Gut tissue was incubated in 30mM EDTA-containing PBS for 13 minutes at room temperature then immediately washed with EDTA-free cold PBS to remove epithelium.  Washed gut specimens were incubated in DMEM containing 1 mg/mL Collagenase P , 0.2 mg/mL Liberase TH and 0.33 mg/mL DNase I  at 37ºC. Dissociated cells were collected every 30 min and strained with a 100 μm nylon mesh, and then were subjected to density gradient centrifugation using 40%/80% Percoll system (Cyvita). The cells at the intermediate layer were collected by centrifuge and resuspended in 2% FBS/2 mM EDTA/PBS. The isolated cells were stained with antibodies against PDPN, CD45 and CD31. Fixable viability dye (eBioscience) was used to stain dead cells. Alive PDPN+CD45-CD31- cells were sorted for single cell 3' gene expression library construction. Library construction was performed according to the manufacter's instructions (single cell 3' v3.1 protocol, 10x Genomics) with slight modification.  Cells up to 10,000 were directly sorted into the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). .