Fresh tumor samples from xenografts were flash-frozen and maintaned at -80 degrees C.
Extracted molecule
genomic DNA
Extraction protocol
Biopsies were minced in the presence of NST buffer and DAPI according to published protocols (Ruiz et al, 2011). Nuclei were disaggregated then filtered through a 40 μm mesh prior to flow sorting with an Influx cytometer (Becton-Dickinson, San Jose, CA) with ultraviolet excitation and DAPI emission collected at >450 nm. DNA content and cell cycle were analyzed using the software program MultiCycle (Phoenix Flow Systems, San Diego, CA).DNAs were extracted using Qiagen micro kits (Qiagen Valencia, CA).
Label
Cy3
Label protocol
DNAs from frozen tissue were treated with DNAse 1 prior to Klenow-based labeling. High molecular weight templates were digested for 30 minutes. In each case 1 ul of 10x DNase 1 reaction buffer and 2 μl of DNase 1 dilution buffer were added to 7 μl of DNA sample and incubated at room temperature then transferred to 70°C for 30 minutes to deactivate DNase 1. Sample and reference templates were then labeled with Cy-5 dUTP and Cy-3 dUTP respectively using a BioPrime labeling kit (Invitrogen, Carlsbad, CA) according to our published protocols (Ruiz, C., et al., Proc Natl Acad Sci U S A, 2011. 108(29): p. 12054-9). All labeling reactions were assessed using a Nanodrop assay (Nanodrop, Wilmington, DE) prior to mixing and hybridization to CGH arrays (Agilent Technologies, Santa Clara, CA).
Fresh tumor samples from xenografts were flash-frozen and maintaned at -80 degrees C.
Extracted molecule
genomic DNA
Extraction protocol
Biopsies were minced in the presence of NST buffer and DAPI according to published protocols (Ruiz et al, 2011). Nuclei were disaggregated then filtered through a 40 μm mesh prior to flow sorting with an Influx cytometer (Becton-Dickinson, San Jose, CA) with ultraviolet excitation and DAPI emission collected at >450 nm. DNA content and cell cycle were analyzed using the software program MultiCycle (Phoenix Flow Systems, San Diego, CA).DNAs were extracted using Qiagen micro kits (Qiagen Valencia, CA).
Label
Cy5
Label protocol
DNAs from frozen tissue were treated with DNAse 1 prior to Klenow-based labeling. High molecular weight templates were digested for 30 minutes. In each case 1 ul of 10x DNase 1 reaction buffer and 2 μl of DNase 1 dilution buffer were added to 7 μl of DNA sample and incubated at room temperature then transferred to 70°C for 30 minutes to deactivate DNase 1. Sample and reference templates were then labeled with Cy-5 dUTP and Cy-3 dUTP respectively using a BioPrime labeling kit (Invitrogen, Carlsbad, CA) according to our published protocols (Ruiz, C., et al., Proc Natl Acad Sci U S A, 2011. 108(29): p. 12054-9). All labeling reactions were assessed using a Nanodrop assay (Nanodrop, Wilmington, DE) prior to mixing and hybridization to CGH arrays (Agilent Technologies, Santa Clara, CA).
Hybridization protocol
Labeled DNA was hybridized without ozone scavenger and chips washed according to manufacture's protocol for 40 hours in a rotating 65°C oven.
Scan protocol
All microarray slides were scanned using an Agilent 2565C DNA scanner and the images were analyzed with Agilent Feature Extraction version 11.0 using default settings.
Data processing
Data was extracted from the TIFF files using Agilent FE 11. The aCGH data was assessed with a series of QC metrics then analyzed using an aberration detection algorithm (ADM2) (Lipson et al., J Comput Biol. 2006 Mar;13(2):215-28). The latter identifies all aberrant intervals in a given sample with consistently high or low log ratios based on the statistical score derived from the average normalized log ratios of all probes in the genomic interval multiplied by the square root of the number of these probes. This score represents the deviation of the average of the normalized log ratios from its expected value of zero and is proportional to the height h (absolute average log ratio) of the genomic interval, and to the square root of the number of probes in the interval.
