FACSort: For sorting cells expressing AC133, the cells were removed from the culture dish with 0.05% trypsin and 0.02% EDTA (Invitrogen, Carlsbad, CA), washed in PBS containing 1% FCS, stained with CD133-PE antibody (AC133, Miltenyi Biotec, Auburn, CA ) and resuspended at 106 cells per ml in the same buffer. The cells were filtered through a 35 um nylon filter prior to FACSort. Sorting was performed on a FACSDiVa flow cytometer (Becton Dickinson). Side and forward scatter profiles and propidium iodide (PI) staining were used to eliminate cell doublets and dead cells. The 10% of the cells that expressed the highest AC133 fluorescence and the AC133 negative cells were collected. Cells were recovered in 15ml conical plastic tubes containing 1ml of 100% FCS, and an aliquot was removed at the end of the sort and reanalyzed to evaluate purity.
Growth protocol
Caco2: The human colon epithelial cancer cell line Caco2 was obtained from American Type Culture Collection (ATCC, Manassas, VA). Caco2 cells at passage 10 were infected with a Lentivirus reporter vectors that contain the mouse Melk promoter driving enhancer green fluorescent protein (MELK-GFP). Individual clones were isolated with the use of glass cloning rings. Two of these clones were used for further experiments. All cells were cultured in DMEM (Mediatech Inc, Manassas, VA) supplemented with 20% fetal bovine serum (Tissue Culture Biologicals, Tulare, CA) and 37 ug/ml Gentamicin (Sigma-Aldrich, St. Louis, MO). WM115: The melanoma cell line WM115, provided by Boris Fichtman and Zeev Ronai (Burnham Institute for Medical Research, La Jolla, CA), was cultured in 10% FCS in RPMI 1640. In all experiments, cells were cultured in culture dishes (Nunc) at 37ºC in a humidified 5% CO2 incubator.
Extracted molecule
total RNA
Extraction protocol
Total RNA from AC133 high and negative sorted Caco2 and WM115 cells was extracted using the TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer’s protocol. Two samples each of two cell lines were analyzed as biological duplicates. Labeled cRNA was prepared from 500 ng RNA using the Illumina® RNA Amplification Kit from Ambion (Austin, TX, USA). The Biotin labeled cRNA (750 ng) was hybridized 18 hr at 58ºC to the HumanRef-8 v2 Expression BeadChip (>22,000 gene transcripts; Illumina, San Diego, CA, USA) according to the manufacturer’s instructions.
Label
Streptavidin Cy3
Label protocol
As described in the illumina Human-8 chip processing protocol. Briefly, generation of biotin labelled cRNA from the product of RT cDNA synthesis and 2nd strand cDNA synthesis.
Hybridization protocol
As recommended by illumina.
Scan protocol
As suggested by illumina using the illumina scanner.
