Illumina HiSeq 2500 paired end sequencing; Transcriptomic profiling of human purified intestinal epithelial samples and organoids from paediatric and foetal small and large intestine
Transcriptomic profiling of human purified intestinal epithelial samples and organoids from paediatric and foetal small and large intestine
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.  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
Experimental Factor: organoids
biosource type
Experimental Factor: juvenile stage
developmental stage
Experimental Factor: terminal ileum
sampling site
Experimental Factor: 2
block