Illumina HiSeq 2500 paired end sequencing; Whole-exome sequencing (WES) to assess genomic concordance between an adenoma/adenocarcinoma colonoid culture 14-881 and the source patient tissue
Whole-exome sequencing (WES) to assess genomic concordance between an adenoma/adenocarcinoma colonoid culture 14-881 and the source patient tissue
Gastrointestinal tissue was collected as part of a surgical resection through the University of Michigan Tissue Procurement Core and was approved by the University of Michigan Medical School Institutional Review Board with informed consent obtained from the patient. Adenoma tissue, associated with invasive moderately differentiated colorectal adenocarcinoma, and histologically normal margin tissue were collected. Colonic adenoma epithelium organoid cultures were established and maintained as according to Dame et al. (Dame et al. Development. 2018; Dame et al., Lab Invest. 2014). To establish colonoids, the mucosa was separated from resected surgical tissue and incubated with 10 mM dithiothreitol (Sigma-Aldrich) for 15 minutes prior to cold incubation with 8 mM ethylenediaminetetraacetic acid (Sigma-Aldrich) for 75 minutes. Epithelial fragments were detached from the lamina propria by snap-shaking, washed three times in cold DPBS, and seeded into 8 mg/mL Matrigel (Corning #354234) on 6-well tissue culture plates (USA Scientific CytoOne, #CC7682-7506). The growth medium consisted of KGM-Gold™, (#195769, Lonza), a serum-free epithelial medium containing a reduced calcium concentration of 0.15mM (with CaCl2; PromoCell GmbH; #34006), with manufacturer proprietary concentrations of hydrocortisone, epinephrine, pituitary extract, epidermal growth factor, insulin and transferrin, and including the antimicrobials 25μg/mL gentamicin (Gibco, #15750060) and 50μg/mL Primocin (InvivoGen, #ant-pm-1). Cultures were supplemented with 10 µM Y27632 (Miltenyi Biotec GmbH, #130-104-169) for the first 18 days of establishment. Medium changes occurred daily. Cultures were split every 5-7 days using mechanical dissociation from previous Matrigel by a 1 mL pipet, followed by re-plating in fresh Matrigel in 10 µL droplets. All plasticware, excluding culture plates, were coated in 0.1% bovine serum albumen in DPBS (Sigma-Aldrich; A8806) to prevent tissue/colonoids from adhering to surfaces. Cultures were confirmed to be negative for mycoplasma and were tested for the presence of viruses (CLEAR with Infectious Disease PCR Panel, Charles River Laboratories). Lines were authenticated to match source tissues by short tandem repeat analysis at the University of Michigan DNA Sequencing Core.  Colonic adenoma epithelium organoids were grown in a reduced serum-free growth media, KGM-Gold™, and harvested for DNA isolation at 2 months and 6.5 months in culture.  Organoids were isolated from Matrigel droplets by mechanical disruption of the Matrigel with a 200 μL pipet tip in cold DPBS, rotated for 15 minutes at 4°C in 2 mM EDTA, and washed three times in cold DPBS with 3-minute centrifugations at 250xg, 4°C. DNA was extracted from tissue specimens first preserved in Allprotect Tissue Reagent (Qiagen, #76405) or directly from fresh colonoid preparations with the All prep DNA/RNA/protein mini kit (Qiagen, #80004). DNA was fragmented to 250bp using standard Covaris sonication. Fragmented DNA was prepared as a standard Illumina gDNAlibrary using IntegenX reagents. The samples were PCR amplified and the libraries were checked for quality and quantity.