Single-Cell Transcriptome Analysis of MAIT Cell Subsets from human normal intestine
Single-Cell Transcriptome Analysis of MAIT Cell Subsets from human normal intestine
De-identified intestinal tissues were collected. Relative normal tissues from patients with colorectal cancer were obtained and stored in cold SPS-1 storage buffer before immediate processing.  For isolation of lymphocytes from intestines, intestine tissues were cut into small pieces, and epithelial cells were dissociated with HBSS containing 5 mM EDTA, 1 mM DTT, and 2% FBS, shaken for 20 minutes at 37°C. This process was performed twice. After each step, samples were vortexed, rinsed with PBS, and the epithelial cell layer was discarded. The remaining tissue was digested in HBSS with 1 mg/mL Liberase TM, and 20 μg/mL DNase I for 30 minutes at 37°C. Percoll centrifugation was performed to remove dead cells and debris. The resulting single cells were strained through a 70 μM mesh.  MAIT cells were sorted by fluorescence activated cell sorting (FACS) after surface marker staining. cDNA-seq libraries were prepared using the chromium 5' single cell gene expression kit from 10x genomics according to the manufacturer's instructions.