title: Single Cell Integration Characterises Gastric Cell Metaplasia in Inflammatory Intestinal Diseases
design: Single Cell Integration Characterises Gastric Cell Metaplasia in Inflammatory Intestinal Diseases
library_construction_protocol: Donors were perfused with cold University of Wisconsin (UW) solution and fresh tissue was collected from the distal stomach (antrum/pylorus), duodenum and terminal ileum within 1h of circulatory arrest, and stored in HypoThermosol FRS preservation solution (Sigma, H4416) at 4°C until processing. Intestinal tissue was open longitudinally and rinsed with D-PBS and then processed to single-cell suspensions following standard protocols. For tissues from donor A68/759B (D105) epithelium and lamina propria (LP) were separated into different fractions by dissection. Epithelial cells were removed by washing the intestinal mucosa twice in Hank's Balanced Salt Solution (HBSS) medium (Sigma-Aldrich) containing 5 mM EDTA (Thermo Fisher, 15575020), 10 mM HEPES (Gibco, 42401042), 2% (v/v) FCS supplemented with 10 mM ROCK inhibitor (Y-27632 (Merck, Y0503)) while shaking at 4°C for 20 minutes. Epithelial wash-offs were centrifuged at 300 g for 7 min at 4°C and incubated at 37°C with TrypLE (Thermo Fisher) supplemented with 0.1 mg/mL DNase I (Sigma, 11284932001) for 5 minutes. Cells were pelleted and filtered through a 40 μm cell strainer and resuspended in Advanced DMEM F12 (ThermoFisher, 12634028) with 10% (v/v) FCS. The remaining epithelium-depleted tissue was minced and incubated in digestion media (HBSS medium, 0.25 mg/mL Liberase TL (Roche, 5401020001) and 0.1 mg/mL DNase I (Sigma, 11284932001)) on a shaker at 37°C for up to 45 min. The tissue was gently homogenised using a P1000 pipette every 15 mins.  Donors were perfused with cold University of Wisconsin (UW) solution and fresh tissue was collected from the distal stomach (antrum/pylorus), duodenum and terminal ileum within 1h of circulatory arrest, and stored in HypoThermosol FRS preservation solution (Sigma, H4416) at 4°C until processing. Intestinal tissue was open longitudinally and rinsed with D-PBS and then processed to single-cell suspensions following standard protocols. For tissues from donor A68/770C (D99), full thickness tissue was diced with a scalpel and digested in digestion media (HBSS medium, 0.25 mg/mL Liberase TL (Roche, 5401020001) and 0.1 mg/mL DNase I (Sigma, 11284932001)) on a shaker at 37°C for up to 45 min. Cells were pelleted and filtered through a 70 μm strainer before proceeding to Chromium 10x Genomics single cell 5' v2 protocol as per manufacturer's instructions.  For samples from donors PT-61, PT-62, PT-64 and PTG65, uninvolved tissue from preterm infants, between 23 and 31 post conception weeks (pcw), with necrotising enterocolitis (NEC), focal intestinal perforation or intestinal fistula (n = 4) were collected. Tissue was resected from the infant and placed immediately into ice cold PBS. Within 3 hours, samples were enzymatically dissociated into a single cell suspension using Collagenase Type IV (Worthington) for 30 minutes at 37°C. Cells were filtered with 100 µm cell strainer, treated with red blood cell lysis and filtered through a 35 µm strainer. Cells were stained with DAPI before FACS sorting, selecting only for live, single cells and separating CD45 positive and negative cells. Sorted cells were then loaded onto the Chromium Controller (10x Genomics) using the Single Cell Immune Profiling kits and subsequently sequenced as per the manufacturer's protocol. Cells were loaded into the 10x Chromium Controller according to the manufacturer's protocol at a concentration that would attain between 8,000-10,000 cells/chip position. cDNA library preparation was were carried out according to the manufacturer's protocol.