Illumina HiSeq 4000 sequencing; VDJ: Single-cell RNA sequencing of human steady-state immune cells from the lamina propria of colon and matching mesenteric lymph nodes
VDJ: Single-cell RNA sequencing of human steady-state immune cells from the lamina propria of colon and matching mesenteric lymph nodes
Human tissue was obtained from deceased transplant organ donors after ethical approval (reference 15/EE/0152, East of England Cambridge South Research Ethics Committee) and informed consent from the donor family. Fresh mucosal tissue from the caecum, transverse colon and sigmoid colon, and lymph nodes from the intestine mesentery, were excised within 60 minutes of circulatory arrest and colon tissue preserved in University of Wisconsin (UW) organ preservation solution (Belzer UW® Cold Storage Solution, Bridge to Life, USA) and mLN stored in saline at 4°C until processing. Tissues from donor 390c were manually diced and <5.0g was added per miltenyi C tube with 5mL tissue dissociation media (1x Liberase TL (0.13U/mL/DNase; Roche) and Benzonase nuclease (10U/mL; Merck) in 1% FCS and 20mM HEPES in PBS (Lonza). Samples were dissociated with GentleMACS Octo for 30 minutes homogenising/37oC cycle. Enzymatic digestion was stopped with the addition of 2mM EDTA in tissue dissociation media. Digested samples were then passed through a 70uM smart strainer (Miltenyi) before being washed with PBS and pelleted at 500g for 10 minutes. Cells were resuspended in PBS, layered onto FicollPaque Plus (GE Healthcare) and spun at RT 400g for 25 minutes. Mononuclear cells were retrieved from ficoll layer and washed with PBS. Cells were filtered through 0.2uM filter (FLowmi cell strainers, BelArt). Cells were manually counted using a haemocytometer and diluted to a concentration of 1000 cells/µl in 0.04% BSA in PBS.  Cells were loaded into the 10x Chromium Controller according to the manufacturer's protocol at a concentration that would attain between 2000-5000 cells/chip position. 10x Genomics VDJ libraries were generated from the 5' 10x Genomics Chromium cDNA libraries as detailed in the manufacturer's protocol.
Wellcome Sanger Institute
E-MTAB-8476:Pan_T7935498_p