# characteristics.INSDC center name[].text
University of Cambridge Department of Paediatrics

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Namespace:University of Cambridge Department of Paediatrics

# characteristics.description[].text
Protocols: Following ethical approval (REC-12/EE/0482) and informed consent, intestinal biopsies were collected from the terminal ileum (TI) and sigmoid colon (SC) from children under 16 years of age undergoing routine diagnostic endoscopy. All children included in this study were found to have macroscopically and histologically entirely normal mucosa as well as no subsequent evidence of any underlying GI pathology. Foetal intestine was obtained with ethical approval (REC-96/085) and informed consent from elective terminations for reasons other than maternal physical health or foetal disease at 8-12 week gestation. Foetal gut was dissected and divided into the proximal (small intestine) and distal (large intestine) sections at the ileo-caecal junction. Purification of intestinal epithelial cells Samples were washed three times in Hank’s Balanced Salt Solution (HBSS), followed by enzyme digestion using a mix of liberase and hyaluronidase in HBSS for 45 min at 37 °C. The cell suspension was passed through a 40 μM sieve, spun down at 1500 rpm at 4 oC for 7 min and re-suspended in phosphate-buffered saline (PBS) containing 10 % bovine serum albumin (BSA) and 100 IU/ml DNase. Cells were then stained with anti-CD326 magnetic microbeads and Fc-Receptor block for 30 min at 4 oC. A magnetic-associated, automated cell separation system (MACS, Miltenyi Biotec) was used to positively select for CD326+ cells. Sorted cells were re-suspended in RLT Plus lysis buffer (Qiagen) supplemented with 1 % β-Mercaptoethanol, passed through a Qiagen shredder column and stored at -80 °C until further processing. Mucosal biopsies were washed three times with cold, sterile PBS. For foetal organoids, the dissected small or large intestine was washed and minced with a scalpel blade. Tissues were incubated for 30 min at 4o C on a roller- mixer in 2.5 mM EDTA solution. EDTA was dissolved in either Complete Chelating Solution + Dithiothreitol (5.6 mM Na2HPO4, 8.0 mM K2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM Sucrose, 54.9 mM D-Sorbitol, 0.5 mM DTT, all from Sigma, St. Louis, MO, USA) for TI biopsies and foetal gut or dissolved in PBS for SC biopsies. After five washes with cold PBS, intestinal crypts were released by pipetting. Crypt suspension was spun down at 800g for 5 min and resuspended in Matrigel matrix (Sigma, St. Louis, MO, USA). Around 100 crypts were seeded per well in 20 μl of Matrigel (Corning, Corning, NY USA) on a pre-warmed 48-well plate (Corning, Corning, NY USA) and covered with organoid culture medium DNA and RNA were extracted simultaneously from the same sample using Qiagen AllPrep Mini Kit (Qiagen) Illumina TruSeq protocol according to manufacturer's recommendation