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. 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.