organism: Homo sapiens
description: Protocols: 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). RNA was extracted from sorted cells using the RNeasy Micro Kit (Qiagen; 74004) with on column DNase digestion. RNA concentration and quality were determined using a Nanodrop (Thermo Scientific) and Bioanalyzer (Agilent). ). Due to the small number of cells following FACS sorting and corresponding low level of input RNA, we depleted ribosomal RNAs with RiboGone (Clontech; 634846) and prepared sequencing libraries utilizing the SMARTer Stranded RNA-Seq kit (Clontech) following the manufacturer's recommended protocol.
attributes.tag: ENA first public
attributes.value: 2017-04-27
attributes.tag: ENA last update
attributes.value: 2016-05-17
attributes.tag: External Id
attributes.value: SAMEA3992698
attributes.tag: INSDC center alias
attributes.value: University of Michigan
attributes.tag: INSDC center name
attributes.value: University of Michigan
attributes.tag: INSDC first public
attributes.value: 2017-04-27T17:01:54Z
attributes.tag: INSDC last update
attributes.value: 2016-05-17T15:37:36Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-4698:227_SortedPos
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: colon adenoma organoid cell
attributes.tag: common name
attributes.value: human
attributes.tag: individual
attributes.value: 227
attributes.tag: phenotype
attributes.value: SortedPos (Magnet bound, LGR5 positive by FACS)
attributes.tag: sample name
attributes.value: E-MTAB-4698:227_SortedPos