Single cell sequencing of mucosal pinch biopsies from patients with ulcerative colitis and primary sclerosing cholangitis concomitant with colitis
Single cell sequencing of mucosal pinch biopsies from patients with ulcerative colitis and primary sclerosing cholangitis concomitant with colitis
All patients were recruited as part of the Australian IBD Microbiome (AIM) study. All procedures were covered by ethics approval from South Eastern Sydney Local Health District Research Ethics Committee (HREC ref no:18/173) and followed patient consent. Mucosal pinch biopsies were collected from the ascending colon, transverse colon, descending colon and rectum of patients with either UC or PSC-UC (classified as resembling UC at the clinical level) during routine endoscopy procedures and immediately transferred to ice-cold RPMI medium containing 10% FBS. Inflammatory state was recorded by treating gastroenterologists during endoscopy and histologically confirmed by anatomical pathologists with subspecialty interest in gastrointestinal pathology. Biopsies from each region were processed separately until pooled for capture. Biopsies were washed in 1x PBS before incubation in 1.5 mL digestion medium (1.16 mL HBSS, 246.9 µL liberase DH, 60 µL hyaluronidase, 36 µL DNase I) in 15 mL tubes with rotation at 600 rpm for 25 minutes at 37°C. Biopsies were homogenised at 10 minutes via trituration and vortexing. Digestions were halted by adding 5 mL ice-cold neutralisation medium (20% FBS in RPMI) and cell suspensions passed through 40 µM cell strainers. Digestion tubes were washed with a further 5 mL neutralisation medium passed through the same strainer to transfer any remaining cells. Cell suspensions were then centrifuged at 300g for 10 minutes at 4°C. Supernatants were discarded and cells resuspended in 1 mL resuspension medium (1% FBS in PBS) and centrifuged again as before. Cells of the right colon (ascending and transverse colon) and left colon (descending colon and rectum) were pooled separately, with hashtag oligos used for in silico deconvolution of discrete anatomical regions for each donor. Supernatants were then discarded and cell pellets resuspended in 45 µL Cell Staining Buffer with 5 µL TruStain Fc Blocking Reagent and incubated for 10 minutes at 4°C to prepare for hashing. TotalSeq C0251 anti-human hashtag 1 and C0252 anti-human hashtag 2 antibody pools were prepared as per the manufacturer's instructions and added to 50 µL blocked cell suspensions and incubated for 30 minutes at 4°C. Hashed cell suspensions were then washed three times by centrifugation at 400 g at 4°C for 5 minutes with 3 mL Cell Staining Buffer. Cell pellets were then resuspended in 100 µL and counted. Regions were pooled equally for two captures: right colon (containing ascending and transverse colon) and left colon (containing descending and rectum). Demultiplexing based on antibody hashtagging was therefore used for higher-precision mapping to each region (e.g. descending colon vs. rectum). For PSC-2 and PSC-3, regions were pooled across 4 captures and cells demultiplexed using SNP information.  Two samples were pooled equally and loaded into the 10x Genomics Chromium Controller according to the manufacturer's protocol at a concentration that would attain between 12,000 cells/chip position. GEX cDNA library preparation was carried out according to the 10x Genomics Chromium Single Cell 5' Reagent Kits User Guide.