The Human Myeloma Cell Lines were obtained from DMSZ-German collection of Microorganisms and Cell Culture, Germany (NCI-H929, OPM2, JJN3, RPMI-8226, and KMS-12); or kindly provided by Dr. T. Otsuki, Kawasaki Medical School, Okayama, Japan (KMS-28, KMS-34, KMS-18, KMS-11, KMS-26, KMS-27, KMM-1 and KMS-20); Dr S. Iida, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan (KM4, FR4, and AMO1), and Dr. F. Malavasi, Department of Genetics, University of Torino, Italy (LP-1); or were established in our laboratory (CMA-01, CMA-02 and CMA-03) [Verdelli D et al.; Haematologica 2005, 90: 1541-1548]. They were cultured in Iscove’s modified Dulbecco’s medium supplemented with 10% fetal calf serum at concentrations ranging from 3×105 to 8×105, at 37°C in a 5% CO2 humidified atmosphere. CMA-01, CMA-02 and CMA-03 were cultured in presence of 20U/ml recombinant human IL-6 (R&D System, Minneapolis, MN, USA).
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA extraction was performed using Wizard® Genomic DNA Purification kit according to the manufacturer's instructions (Promega).
Label
biotin
Label protocol
Biotinylated DNA were prepared according to the standard Affymetrix protocol starting from 250 nanograms of genomic DNA.
Hybridization protocol
Following fragmentation, 90 micrograms of biotin-labeled DNA were hybridized for 16 hr at 49°C on GeneChip® Human Mapping 250K NspI Arrays. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
Scan protocol
Human Mapping 250K NspI arrays were scanned using the GeneChip Scanner 3000 7G (Affymetrix).
Description
Genome-wide profiling data from Human Myeloma Cell Line KM4
Data processing
Genome-wide DNA profiling was performed on Affymetrix GeneChip® Human Mapping 250k Nsp arrays. To find the corresponding CN values, we firstly extracted the raw data from the CEL files using the Affymetrix packages GTYPE 4.1 and Copy Number Analysis Tool 4.0.1 (Affymetrix, Santa Clara, CA, USA) and the Mapping Array 250k Nsp probe annotations released on July, 12 2007. In order to keep only the raw data and thus to avoid the CN inference facility of the latter software package, the Hidden Markov Model Genomic Smoothing window was set to 0. After the preprocessing, piecewise constant estimates of the underlying local DNA CN variation was calculated using the DNA copy Bioconductor package, which looks for optimal breakpoints using circular binary segmentation (CBS).
The VALUE column described as "HmmMedianLog2Ratio - Genomic Bandwith Smoothing 0 Mb" is the result displayed by CNAT4.0.1 software (Affymetrix) when applying Hidden Markov Model algorithm. The Log2Ratio is the base-2 logarithm applied to the ratio between the CEL-extracted probe values in our samples and in a dataset of 48 normal samples belonging to HapMap Project and freely available on Affymetrix website http://www.affymetrix.com/support/technical/sample_data/500k_data.affx. The Genomic Smoothing Window was set to zero in order to extract raw data.
