title: Illumina HiSeq 2500 paired end sequencing; RNA-seq of LGR5(+) and LGR5(-) cells isolated from human colon adenoma organoids and targeted DNA-seq of a colorectal biobank
design: RNA-seq of LGR5(+) and LGR5(-) cells isolated from human colon adenoma organoids and targeted DNA-seq of a colorectal biobank
library_construction_protocol: Tissue was collected from subjects recruited through the Molecular Pathology and Biosample Core (MPBC) as part of the GI SPORE at the University of Michigan. Colonic tissue was acquired by endoscopy (adenomas) or from colonic resections (adenocarcinomas) and according to protocols approved by the University of Michigan Institutional Review Board (IRB; HUM00064405/0038437/00030020). Two growth media were used; the first for continuous culture and the second to drive to a cyst morphology. The first reduced medium (KGM-Gold) is a serum-free epithelial medium containing epidermal growth factor (EGF) and pituitary extract (KGMG; Lonza; 195769) with the antimicrobials 25µg/mL gentamicin (Invitrogen; 15750) and 50µg/mL Primocin (InvivoGen; ant-pm-1) and made to 0.15mM calcium with CaCl2 (PromCell). The second (L-WRN medium) contains high levels of Wnt3a, R-spondin-3 and Noggin provided by a conditioned medium from support L- cells as described by Miyoshi 3. The complete L-WRN medium further contained Advanced DMEM/F-12, 2mM GlutaMax, 10mM HEPES, N-2 media supplement, B-27 supplement minus vitamin A, 100 units/mL penicillin, 0.1 mg/mL streptomycin (all Invitrogen), 50µg/ml Primocin, 1mM N-Acetyl-L-cysteine (Sigma; A9165), and 100ng EGF/mL (R&D; 236-EG), as well as the addition of 10mM Nicotinamide (Sigma; N0636). To drive from a budding to cyst morphology, cultures are gradually switched from KGMG to L-WRN for three to four weeks (3 days KGMG, 5 days 50/50 L-WRN; 5 days 75/25; 100% L-WRN until predominately cystic). Consistent with previous reports, organoids formed cystic structures in the presence of Wnt ligand provided by L-WRN medium 4-8. Cultures were propagated in Matrigel (Corning; 354234) which was made to 8mg/mL in growth media on 6-well tissue culture plates (USA Scientific CytoOne; CC7682-7506). All plasticware, excluding culture plates, were coated in 0.1% bovine serum albumen in DPBS (Sigma; A8806) to prevent tissue/organoids/cells from adhering to surfaces. Cultures were passaged every 4-7 days by digesting Matrigel in cold 2mM EDTA in DPBS and plated the first day with the Rho-associated protein kinase (ROCK) inhibitor, Y27632 (10µM; Miltenyi; 130-104-169). On the day of sorting, the cultures were treated with 10µM Y27632 for 2.5 hrs prior to harvest. Matrigel was digested for 45 minutes with cold 2mM EDTA in DPBS and organoids were washed 3X with cold DPBS (100xg at 4C). Structures were dissociated into single cells using the Tumor Dissociation Kit (human) (Miltenyi Biotec; 130-095-929) with the protocol modifications described as follows. The enzymes were prepared in warm HBSS modified to 0.13mM calcium and 0.9mM magnesium [10% HBSS, calcium, magnesium, no phenol red (Life Technologies 14025092) with 90% HBSS, no calcium, no magnesium (Life Technologies 14170112)] to minimize differentiation of the epithelial cells while supporting enzymatic activity. The organoids were suspended in 20mL of enzyme solution containing 5µM Y27632, then transferred to two BSA-coated C-tubes (Miltenyi Biotec; 130-096-334) and dissociated with a gentleMACS Dissociator (Miltenyi; 130-093-235) using the program h_tumor_01, once at the onset and every 15 minutes for 1 hour, and finishing with two program runs. Between runs the suspension was slowly rotated at 37C. The cell suspension was then poured over a succession of cold BSA-coated cell strainers into 0.5% BSA-2mM EDTA-PBS: 100µm (Corning; DL 352360) to 40µm (Corning; DL 352340) to 20µm (CellTrics; 04-0042-2315). The cells were washed 3X in the 0.5% BSA-2mM EDTA-PBS and centrifuged for 5 minutes at 500xg. Cells were counted under trypan blue exclusion to estimate live cell number using a Countess Automated Cell Counter (Invitrogen; C10227).  DNA was extracted from whole tissue or organoid cultures using the DNeasy Mini Kit (Qiagen). DNA concentration and quality were determined using a Nanodrop (Thermo Scientific) and Bioanalyzer (Agilent). Targeted panel for colorectal cancer. Targets 71 genes.
attributes.tag: Experimental Factor: individual
attributes.value: 590
attributes.tag: Experimental Factor: disease
attributes.value: adenomatous colon polyp
attributes.tag: Experimental Factor: growth condition
attributes.value: organoid culture; KGM medium
attributes.tag: Experimental Factor: protocol
attributes.value: DNA-seq