GSM8078413: Ileum, Crohn's disease, Non-inflamed, rep 2, scRNA-seq; Homo sapiens; RNA-Seq
Two pieces of matched ileum and the associated mesentery was collected from consenting patients who were undergoing surgical resection of their ileum to treat Crohn's disease. Under a pathologist's supervision, one piece of tissue was collected from an area of the ileum with active inflammation and the other piece was from adjoining normal region. Control ileum and mesentery was collected from gastrointestinal cancer patients from regions of the intestine upstream of their tumors. After collection, tissue was stored for under an hour in RPMI-1640 before undergoing single cell digestion. In the lab, the tissue was first rinsed in fresh HBSS before the mesentery, intestinal lamina propria, and muscularis were separated using a scalpel. To remove mucus and epithelial cells, the lamina propria was cut into thin 0.5 cm thick strips and then placed in 10 ml of HBSS with 5 mM EDTA and digested at 37° C for 20 minutes, before vortexing the tubes for 20 seconds, removing the tissue from the HBSS/ETDA, placing it in fresh HBSS and vortexing the tissue again. The lamina propria was then minced into small pieces and weighed. Mesentery was also minced into small pieces with a scalpel, before being weighed. Then tissue was digested in 1 g of tissue per 5 ml of RPMI-1640 media containing 10% FBS, 1 mg/ml Collagenase IV, 0.2 mg/ml DNase I at 37° C for 45 minutes (for mesentery) or 1 hour (for ileum) on an orbital at 100 rpm. Tissue was then vortexed, filtered through a 100m filter into a fresh tube, and washed with an equal volume of FACS buffer (2% FBS, 5mM EDTA, 0.2% sodium azide in PBS). Cells were then spun down for 10 minutes at 500 x g at 4° C, RBC lysis was performed for 5 minutes on the cells, which were washed with additional 10 ml of FACS buffer. After spinning cells down at 10 minutes at 500 x g at 4C, they were resuspended in 1 ml of FACS buffer, counted using an automatic cell counter and frozen at -80° C in 10% DMSO in 90% FBS before being moved to long-term storage in liquid nitrogen. On a later day, aliquots of cells were rapidly thawed in a 37° C water bath, stained for flow cytometry sorting and live, CD45+ immune cells were sorted for sequencing. The 10x Genomics Chromium Single Cell 5' Library and Gel Bead Kit was used. We sequenced 5' RNA expression, TCR sequencing, and BCR sequencing as per the manufacturer's protocol.