cell line: ERN2 cell type: primary breast cancer histology: invasive ductal carcinoma estrogen receptor status: negative erbb2 status: not amplified
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was extracted from the cell lines and human tumor tissue with the QIAamp DNA Micro kit (QIAGEN) per manufacturer’s protocol. The genomic DNA used to analyze copy number alterations on the SNP6.0 arrays was processed per manufacterer's instructions. The GAPF assay was performed with 50% formamide as described (Diede et al. 2010a). Two micrograms of genomic DNA from cells were split evenly and digested with 10 U of SbfI, KpnI or PmeI (NEB) for at least eight hours at 37°C in a volume of 20 μL. The restriction enzymes were heat inactivated by incubation at 65°C for 20 minutes. The digests were then combined into one tube. To the mixture was added 3 μL of 3 M NaCl (final concentration of 100 mM), 45 μL of formamide (final concentration of 50%), and 2 μL of water. This mixture was heated to 99°C in a thermocycler for 7 minutes to denature the DNA, and then rapidly cooled in a ice-water bath for 3 minutes leading to the formation of “snap-back” DNA. S1 digestion was performed by adding 100 U of S1 nuclease (2 μL of 100 U/μL solution; Invitrogen), 12 μL of 10x S1 nuclease buffer, and 8 μL of 3 M NaCl. The sample was incubated at 37°C for 60 minutes. S1 nuclease was extracted with phenol:chloroform, and the DNA was ethanol precipitated in the presence of 20 ug glycogen. The DNA pellet was resuspended in 80 μL of 1/10 TE. The DNA was split equally between two tubes and digested with 40 U of MseI (NEB) or 40 U of MspI (NEB) for at least 8 hours at 37°C in a reaction volume of 50 μL. The restriction enzymes were heat inactivated by incubation at 65°C for 20 minutes. For the ligation-mediated PCR, linkers were generated by creating a 100 pM equimolar solution of each linker oligo in 10 mM TE supplemented with NaCl for a final salt concentration of 100 mM. This solution was incubated in a boiling water bath for 7 minutes and then allowed to cool to 25°C to permit annealing. Linkers were recovered by ethanol precipitation and resuspended in 500 μL of water. The linkers were ligated to the MseI- or MspI-digested DNA by adding 5 μL of appropriate linker, 10x T4 DNA ligase buffer, water and 400 U of T4 DNA ligase (NEB) in a 50 μL reaction volume. This reaction was incubated at 16°C for at least 8 hoursm, and the ligase was heat inactivated at 65°C for 10 minutes. Free linkers were removed using a YM-50 spin column (Microcon) by adding the ligation reaction and 160 μL of 1/10 TE to the column and spinning at 12,000xg for 5 minutes or until the membrane is almost dry. The sample was recovered by adding 20 μL of 1/10 TE to the membrane, incubating at room temperature for 5 minutes, and spinning at 1000xg for 3 minutes per manufacturer’s instructions. The PCR was performed with the FastStart High Fidelity PCR System (Roche) with the following reaction reagents: 4 μL of linker-ligated DNA, 10 μL of 10x FastStart PCR buffer, 10 μL of 2mM dNTPs, 12 μL of 10 pM linker-specific primer, 20 μL of 5x GC-rich solution, 1 μL of FastStart Taq Polymerase, and 43 μL of water. The PCR conditions were as follows: 96°C for 6 minutes; 30 cycles of 96°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds; 72°C for 7 minutes. Three microliters of the PCR product were run on a 1.5% agarose gel for quality control. The products shoμLd range from 200 bp to 600 bp. The MseI and MspI PCR products were combined and concentrated in a YM-30 spin column (Microcon). The combined PCR products (~200 μL) and 300 μL of 1/10 TE were added to the membrane and spun at 14,000xg 40 to 60 minutes, checking every 15 minutes until the volume is approximately 25 μL. The DNA was recovered per manufacturer’s instruction. The DNA was quantitated using the NanoDrop system (Thermo Scientific).
Label
biotin
Label protocol
Seven and a half micrograms of DNA were fragmented using 0.016 U of DNaseI (NEB) and 10x DNaseI buffer in a 50 μL reaction. The reaction was incubated at 37°C for 25 minutes followed by heat inactivation at 95°C for 15 minutes. The fragmented samples were enzymatically biotinylated using the GeneChip WT Double-Stranded DNA Terminal Labeling Kit (Affymetrix) per manufacturer’s protocol.
Hybridization protocol
Seven and a half micrograms of biotin-labeled DNA was hybridized to the appropriate Affymetrix Human Tiling 2.0R Array for 16 hours at 45°C at 60 rpm in an Affymetrix hybridization oven per manufacturer’s protocol.
Scan protocol
The arrays were scanned on an Affymetrix Scanner 3700 7G.
Description
ERN2 Copy Number profile, SNP6.0
Data processing
Affymetrix Human Tiling 2.0R Arrays were analyzed using Tiling Array Software (v 1.1.02 Affymetrix). Probe locations were mapped using the NCBI36/hg18 genome build from March 2006. Raw-intensity data were scaled to a target intensity of 100 and normalized by quantile normalization. Normalized probe intensities were analyzed by Wilcoxon rank sum one-sided test, detecting probes with intensities significantly different between the cancer and normal reference samples. The probe analysis for determining signal ratios and p-values was performed using a bandwidth of 500 bp. pvalue.bar and signal.bar files were generated using the Tiling Array Software (Affymetrix) as described in the data processing protocol. The pvalue.bar files contain -10log10 pvalues calculated form a Wilcoxon one-sided test. The signal.bar files contain Log2 signal intensity (test/control).
