Esophageal cancer cell lines (PT-1590 and OE-19) were grown in RPMI 1640 medium (PAN-Biotech) supplemented with 10% fetal calf serum (FCS)(PAN-Biotech), 2mM L-Glutamic acid (PAN-Biotech) and 1% of Penicillin/Streptomycin solution (PEN STREP) (PAN-Biotech). Breast cancer cell lines SKBR3, MDA-MB-453 and MDA-MB-361 were cultured in DMEM (PAN-Biotech) medium supplemented with 10% FCS (PAN-Biotech), 2mM L-Glutamic acid (PAN-Biotech) and 1% PEN STREP (PAN-Biotech). BT-474 was grwon in RPMI 1640 medium supplemented with 10% FCS, 2mM L-Glutamic acid, 1% P/S and 1mM sodium pyruvate.
Extracted molecule
genomic DNA
Extraction protocol
Total genomic DNA (bulk DNA) was extracted using the Dbeasy Blood & Tissue Kit (QIAgen) following manufacture's instructions. All single cells, cell pools (few hundred cells) and microdissected specimens from formalin fixed paraffin embedded (FFPE) tumor tissue were amplified using the Ampli1™ WGA Kit (Silicon Biosystems).
Label
Cy5
Label protocol
Random-primed DNA labeling approach (RP labeling): Test and reference DNA samples were labeled using SureTag DNA Labeling Kit (Agilent Technologies) according to the instruction provided by the supplier (Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis, version 7.1, December 2011). Briefly, 1.5 - 2.0 µg of the purified input DNA (WGA product or unamplified genomic DNA) was supplemented with 5 µl of Random Primer Mix and filled up with H2O to 31 µl. Unamplified DNA and WGA products samples were denatured at 95◦C for 10 or 3 minutes, respectively. Sample tubes were transferred on ice and incubated for 5 min. The labeling reaction with exo-Klenow fragment consisted of the following ingredients: 31 µl of denatured DNA, 10 µl of 5x Reaction Buffer, 5.0 µl of 10x dNTP Mix, 3.0 µl of Cy5-dUTP (test) or Cy3-dUTP (reference) and 1.0 µl of Exo(-) Klenow fragment. Labeling reaction was run at 37◦C for two hours, followed by an inactivation step at 65◦C for 10 minutes. Labeled DNA was purified using Ultra 0.5 purification system with a size cut-off of 30 kDa. DNA yields and dye incorporation rates were quantified using the NanoDrop ND-1000 instrument. PCR-based labeling using dye-conjugated universal primer (PCR-T1): Placement of the dye on the universal primer provides the advantage that all restriction digestion fragments present in the WGA product irrespectively of their size will be labeled with the same amount of dye. To avoid cross-hybridization of adapter sequences flanking amplicons in the WGA products, test and reference samples were labeled using different PCR-adapters. Test samples were labeled with the PCR-adapter incorporated in the Ampli1™ WGA kit, while all the reference DNA samples were amplified using the following adapters: MIB5 (5’-TGAGCTGGTCATTGCGCATGGT-3’) and ddMse XI (5’-TAACCATGCGC-3’). Universal primers used in the labeling reaction were directly conjugated with either Cy5 in the case of Ampli1™ universal primer [5’-TAGTGGGATTCCTGCTGTCAGT-3’] or Cy3 in the cases of MIB5 primer [5’-TGAGCTGGTCATTGCGCATGGT-3’] (underscores indicate the placement of the dye). The labeling PCR was run in a total volume of 50 µl reaction composed of 5 µl of 10x Expanded Long Template Buffer 1 (Roche Diagnostics), 2.4 µM of dye-conjugated LIB1 or MIB5 primer (for test and reference sample, respectively), 350 µM of dNTPs, 0.5 µl BSA (Roche), 3.75 U of Expand-Long-Template DNA Polymerase (Roche Diagnostics) and 1.0 µl of the template DNA. The PCR was programmed as follows: 10 cycle of 94◦C for 15 sec, 51◦C for 30 sec and 65◦C for 3:30 min, 2 cycles of 94◦C for 15 sec, 51◦C for 30 sec and 65◦C for 3:30 min (extended by 10 sec every cycle), followed by a final elongation step of 7 min at 65◦C. Products were purified using the Amicon Ultra 0.5 System (cut-off size 100 kDa) and quantified with the NanoDrop ND-1000 instrument. PCR-based labeling using incorporation of dye-conjugated dNTPs (PCR-T2): WGA products were labeled by PCR in the presence of Cy5 or Cy3 conjugated dCTP and dUTP. Incorporation of the fluorescent dyes using the dNTPs provided the advantage that adapters flanking each amplicon in the WGA product could be removed prior to aCGH hybridization, thereby avoiding unspecific cross-hybridization of test sample with reference DNA and oligonucleotide probes on the array. For each sample two 50µl PCR reactions were run in parallel each comprising 5 µl of 10x Expanded Long Template Buffer 1 (Roche Diagnostics), 2.4 µM of the universal primer (LIB1 or MseLig-21, depending on the adapter sequence incorporated in the WGA product), 350 µM of dATP and dGTP, 315 µM of dCTP and dTTP, 35 µM of Cy5/Cy3-dCTP and Cy5/Cy3-dUTP (GE Healthcare), 0.5 µl of BSA (Roche Diagnostics), 7.5 U of Expand-Long-Template DNA Polymerase (Roche) and 0.5 µl of the template DNA. Labeling products were pooled and purified using the Amicon Ultra 0.5 System (100 kDa cut-off). DNA yields and dye incorporation rates were quantified using the NanoDrop ND-1000 Instrument.
Channel 2
Source name
Healthy donor: cord blood; a pool of 4 single-cell WGA products from the same donor
Esophageal cancer cell lines (PT-1590 and OE-19) were grown in RPMI 1640 medium (PAN-Biotech) supplemented with 10% fetal calf serum (FCS)(PAN-Biotech), 2mM L-Glutamic acid (PAN-Biotech) and 1% of Penicillin/Streptomycin solution (PEN STREP) (PAN-Biotech). Breast cancer cell lines SKBR3, MDA-MB-453 and MDA-MB-361 were cultured in DMEM (PAN-Biotech) medium supplemented with 10% FCS (PAN-Biotech), 2mM L-Glutamic acid (PAN-Biotech) and 1% PEN STREP (PAN-Biotech). BT-474 was grwon in RPMI 1640 medium supplemented with 10% FCS, 2mM L-Glutamic acid, 1% P/S and 1mM sodium pyruvate.
Extracted molecule
genomic DNA
Extraction protocol
Total genomic DNA (bulk DNA) was extracted using the Dbeasy Blood & Tissue Kit (QIAgen) following manufacture's instructions. All single cells, cell pools (few hundred cells) and microdissected specimens from formalin fixed paraffin embedded (FFPE) tumor tissue were amplified using the Ampli1™ WGA Kit (Silicon Biosystems).
Label
Cy3
Label protocol
Random-primed DNA labeling approach (RP labeling): Test and reference DNA samples were labeled using SureTag DNA Labeling Kit (Agilent Technologies) according to the instruction provided by the supplier (Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis, version 7.1, December 2011). Briefly, 1.5 - 2.0 µg of the purified input DNA (WGA product or unamplified genomic DNA) was supplemented with 5 µl of Random Primer Mix and filled up with H2O to 31 µl. Unamplified DNA and WGA products samples were denatured at 95◦C for 10 or 3 minutes, respectively. Sample tubes were transferred on ice and incubated for 5 min. The labeling reaction with exo-Klenow fragment consisted of the following ingredients: 31 µl of denatured DNA, 10 µl of 5x Reaction Buffer, 5.0 µl of 10x dNTP Mix, 3.0 µl of Cy5-dUTP (test) or Cy3-dUTP (reference) and 1.0 µl of Exo(-) Klenow fragment. Labeling reaction was run at 37◦C for two hours, followed by an inactivation step at 65◦C for 10 minutes. Labeled DNA was purified using Ultra 0.5 purification system with a size cut-off of 30 kDa. DNA yields and dye incorporation rates were quantified using the NanoDrop ND-1000 instrument. PCR-based labeling using dye-conjugated universal primer (PCR-T1): Placement of the dye on the universal primer provides the advantage that all restriction digestion fragments present in the WGA product irrespectively of their size will be labeled with the same amount of dye. To avoid cross-hybridization of adapter sequences flanking amplicons in the WGA products, test and reference samples were labeled using different PCR-adapters. Test samples were labeled with the PCR-adapter incorporated in the Ampli1™ WGA kit, while all the reference DNA samples were amplified using the following adapters: MIB5 (5’-TGAGCTGGTCATTGCGCATGGT-3’) and ddMse XI (5’-TAACCATGCGC-3’). Universal primers used in the labeling reaction were directly conjugated with either Cy5 in the case of Ampli1™ universal primer [5’-TAGTGGGATTCCTGCTGTCAGT-3’] or Cy3 in the cases of MIB5 primer [5’-TGAGCTGGTCATTGCGCATGGT-3’] (underscores indicate the placement of the dye). The labeling PCR was run in a total volume of 50 µl reaction composed of 5 µl of 10x Expanded Long Template Buffer 1 (Roche Diagnostics), 2.4 µM of dye-conjugated LIB1 or MIB5 primer (for test and reference sample, respectively), 350 µM of dNTPs, 0.5 µl BSA (Roche), 3.75 U of Expand-Long-Template DNA Polymerase (Roche Diagnostics) and 1.0 µl of the template DNA. The PCR was programmed as follows: 10 cycle of 94◦C for 15 sec, 51◦C for 30 sec and 65◦C for 3:30 min, 2 cycles of 94◦C for 15 sec, 51◦C for 30 sec and 65◦C for 3:30 min (extended by 10 sec every cycle), followed by a final elongation step of 7 min at 65◦C. Products were purified using the Amicon Ultra 0.5 System (cut-off size 100 kDa) and quantified with the NanoDrop ND-1000 instrument. PCR-based labeling using incorporation of dye-conjugated dNTPs (PCR-T2): WGA products were labeled by PCR in the presence of Cy5 or Cy3 conjugated dCTP and dUTP. Incorporation of the fluorescent dyes using the dNTPs provided the advantage that adapters flanking each amplicon in the WGA product could be removed prior to aCGH hybridization, thereby avoiding unspecific cross-hybridization of test sample with reference DNA and oligonucleotide probes on the array. For each sample two 50µl PCR reactions were run in parallel each comprising 5 µl of 10x Expanded Long Template Buffer 1 (Roche Diagnostics), 2.4 µM of the universal primer (LIB1 or MseLig-21, depending on the adapter sequence incorporated in the WGA product), 350 µM of dATP and dGTP, 315 µM of dCTP and dTTP, 35 µM of Cy5/Cy3-dCTP and Cy5/Cy3-dUTP (GE Healthcare), 0.5 µl of BSA (Roche Diagnostics), 7.5 U of Expand-Long-Template DNA Polymerase (Roche) and 0.5 µl of the template DNA. Labeling products were pooled and purified using the Amicon Ultra 0.5 System (100 kDa cut-off). DNA yields and dye incorporation rates were quantified using the NanoDrop ND-1000 Instrument.
Hybridization protocol
Array CGH was performed on oligonucleotide-based SurePrint G3 Human CGH 4x180K microarray slides (design code: 022060) according the protocol provided by the manufacturer (Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis, version 7.1, December 2011). Slight modifications were introduced WGA products processed with the PCR-based labeling approaches. Here, the hybridization mix consisted of 5.0 µg of Cot1-DNA (Roche Diagnostics), 12 µl of 10x Blocking Reagent (Agilent Technologies), 60 µl of 2x Hi RPM Hybridization Buffer, 1% (v/v) of both Tween20 and Igepal and 19 µl of both test and reference DNA. For each hybridization 100 µl of the hybridization mix was applied on the array and hybridized at 65◦C for 24 h. Following the hybridization, the slides were washed sequentially: twice for 2:30 min in Oligo aCGH/ChIP-on-Chip Wash Buffer 1 (Agilent Technologies) at room temperature, twice for 30 sec min in Oligo aCGH/ChIP-on-Chip Wash Buffer 2 (Agilent Technologies) at 37◦C. Washed slides were immersed in acetonitrile to remove all remaining traces of the wash buffers.
Scan protocol
Scanned on an Agilent G2565CA scanner (Dye channel: R+G; Scan resolution: 2.5µm, double pass; Tiff file dynamic range: 16 Bit; Red PMT gain: 100%; Green PMT gain: 100%; No XDR)
Description
Healthy female donor #2, single-cell #1 (10 probes) Raw data file: US94700331_252206026493_S01_CGH_107_Sep09_1_2.txt Healthy female donor 2_single-cell 1_reamplified single-cell WGA_RP labeling sample used to compare the performance of two labeling approaches (PCR-T2 vs. RP-labeling) - Fig. S6C. Additionaly sample was used demonstrate the necessity of applying aberration filters with a minumum of 25 probes per abrrant interval - Fig. S6C.
Data processing
Microarray TIFF image files were processed with the Agilent Genomic Feature Extraction Software (version 10.7.3.1). The resulting text files were imported and analyzed with Agilent Genomic Workbench Software (version 6.5.0.18 Lite). Aberrant regions were recognized using ADM-2 algorithm with threshold set to 6.5. Centralization algorithm was set to a threshold of 6.0 and bin size of 10. To avoid false positive calls, aberration filters were applied to define the minimum log2 ratio (minimum absolute average log ratio for region = 0.3) and the minimum number of probes in an aberrant interval (minimum number of probes in region = 10 for unamplified DNA and freshly picked cells or minimum number of probes in region = 25 for WGA products generated with immunostained cells and FFPE samples). The log2 ratios were obtained using the Agilent Genomic Workbench 6.5.0.18.
