Illumina HiSeq 4000 sequencing; Single-cell RNA sequencing of human steady-state immune cells from the lamina propria of colon and matching mesenteric lymph nodes
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. Two individuals (287C and 296B) had received antibiotics in the week prior to death (Supp. Table 1 of associated paper). Protocol for tissue dissociation can be found here: dx.doi.org/10.17504/protocols.io.tbfeijn. Briefly, 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)/60ug/ml 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. The samples were pelleted and resuspended in 40% Percoll. This was underlayed with 80% Percoll and contributed at 600g for 20 minutes with minimal acceleration and break. Cells at the interface were collected and washed with PBS. Cells were FACS sorted into D-PBS as follows: non-CD4+ T immune cells were sorted as single, zombie aqua-, CD45+, CD3- and CD4-. CD4+ T cells were sorted as single, zombie aqua-, CD45+, CD3+ and CD4+. Sorting was carried out on a BD FACS ARIA Fusion.  FACS sorted cells were manually counted using a haemocytometer and diluted to a concentration of 500 cells/µl in PBS. Cells were loaded according to the manufacturer's protocol for Chromium single cell 3' kit in order to attain between 2000-5000 cells/chip position. The standard protocol for the 10x single cell 3' kit (V2 chemistry) was followed and each cell population loaded onto a separate chip inlet. FACS sorted cells were manually counted using a haemocytometer and diluted to a concentration of 500 cells/µl in PBS. Cells were loaded according to the manufacturer's protocol for Chromium single cell 3' kit in order to attain between 2000-5000 cells/chip position. The standard protocol for the 10x single cell 3' kit (V2 chemistry) was followed and each cell population loaded onto a separate chip inlet.