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. Tissue pieces were manually diced and transferred into 5mM EDTA (Thermo Fisher Scientific)/1mM DTT/10mM HEPES (Thermo Fisher Scientific)/2% FBS in RPMI and incubated in a shaker (~200rpm) for 20 minutes at 37oC. Samples were briefly vortexed before the media renewed and incubation repeated. Tissue pieces were washed with 10mM HEPES in PBS and transferred into 0.42mg/ml Liberase DT (Roche)/0.125 KU DNase1 (Sigma)/10mM HEPES in RPMI and incubation for 30 minutes at 37oC. The digested samples were passed through a 40um strainer and washed through with FBS/PBS. Cells were manually counted using 0.4% Trypan Blue (gibco) and a haemocytometer and diluted to 500 cells/ul 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:Human_colon_16S8157819_p