g2. At the start of cultivation of this study, the PDL of UE6E7T-3 was 60. Further cultures consist of Stage I, Stage II, Stage III, and Stage IV. These four samples were named U3-A, U3-B, U3-C, and U3-DT, respectively. U3-DT cells at Stage IV were deposited into the JCRB bank; the quality and ethics of the cells were verified by the bank. Flow cytometry Cells of Stage I, Stage II, Stage III and Stage IV were harvested with trypsin and suspended in 5% FBS-PBS at the concentrations of 12 x 106 cells/ml. 100 l of each cell suspension was mixed with 5 l of antibody diluted to 20 fold with 5% FBS-PBS. After incubation at 4C for 20 min, cell suspension was washed 2 times with 5% FBS-PBS and was suspended with 500 l of 4% paraformaldehydePBS. Data acquisition of 10,000 cells was performed using FACSCanto. Analysis was performed using FlowJo software. Antibodies used in this test were as follows, APC anti-CD34, FITC anti-CD44, FITC anti-CD45, RPE anti-CD73, FITC anti-CD90, and RPE anti-CD105. APC IgG1, FITC IgG1, RPE IgG1, and RPE IgG3 were also used as negative control. 3 / 23 Alteration in PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19786614 Gene Expression on Transformation Measurement of chromosome number and mFISH analysis Measurement of chromosome number and mFISH analysis were performed as previously described. Briefly, for mFISH analysis, metaphase chromosome spreads and the multicolor probe were denatured with 0.07 N NaOH and hybridized at 37C for 14 d. FISH images were captured and analyzed using a Zeiss Axio Imager microscope and Isis mBAND/mFISH imaging software. Anchorage-independent growth Between 125 and 1105 cells were cultured in POWEREDBY10 medium containing 0.3% agarose on 0.6% agarose for 21 days. Colonies of diameter >300 m were counted. Tumorigenicity assay Tumorigenicity experiments were performed by subcutaneous or intramuscular injection of 1107 U3-DT cells into BALB/cAJcl-nu/nu mice . The protocols were approved by the Laboratory Animal Care and the Use Committee of the National Research Institute for Child and Health Development, Tokyo, Japan. In the fourth week, the transplanted tissues were removed 
and embedded in paraffin blocks. Standard hematoxylin and eosin staining of paraffin-embedded tissue was performed for histological examination of tumorigenicity. cDNA synthesis and purchase PCI32765 whole-transcriptome sequencing After subconfluent culture, 51061107 UE6E7T-3 cells at PDL 80, PDL110, PDL219 and PDL270 were harvested. Each cellular total RNA from the four samples was extracted using QIAGEN RNeasy Mini Kit. RNA samples were stored at -80C until use. Poly-A RNA was used to select mRNA using the SOLiD RiboMinus Kit. Following rRNA depletion, poly-A RNA was used to synthesize cDNA using the SOLiD Whole-Transcriptome Analysis Kit . DNA sequencing was carried out using SOLiD 3 PLUS System as described previously. Mapping sequence data and RNA-Seq analysis SOLiD sequence data were mapped on the NCBI/GenBank Homo sapiens genome sequence. Quality control of these sequence data was performed using the CLC Genomics Workbench-v4.5. At least, 90% length and 80% similarity of the reference gene have been required in order to carry out the mapping of the sequenced reads to reference genome. The Genomics Workbench yields gene expression values in units of “reads per PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19786154 kilobase of exon model per million mapped reads” . All sequence reads have been deposited to the DDBJ database of the National Institute of Genetics, accession number DRA000533. Cluster analysis and pathway analysis Alignment to
