Supplementary Methods
Copy number analysis with HGU133 Plus 2.0
1. Whole Genome Amplification (WGA) by REPLI-g Kit (Qiagen)
1.1 Prepare Solution A, Buffer D1, and Buffer N1 as follows.
(Solution A) Water 450 ml
5M KOH 40 ml
0.5M EDTA (pH 8.0) 10 ml / 500 ml
(Buffer D1) Water 35 ml
Solution A 5 ml / 40 ml (for ≤5 samples)
(Buffer N1) Water 72 ml
Solution B in the kit 8 ml / 80 ml (for ≤5 samples)
1.2 Water 2 ml
Genomic DNA (50 ng/ml in TE0.1) 0.5 ml
Buffer D1 2.5 ml / 5 ml
® prepare 3 tubes per sample, mix and spin down, ® RT x 3 min
1.3 add Buffer N1 5 ml to each tube
® mix and spin down
1.4 prepare the following master mix (90 ml per tube at the step 1.3) in a 0.2 ml tube on ice
Water 64 ml
REPLI-g Buffer, 4x 25 ml
REPLI-g DNA Polymerase 1 ml / 90 ml
1.5 To the above tube, add 10 ml of the genomic DNA of the step 1.3
® mix and spin down
® 30°C x 16 hr
® 65°C x 10 min
® 4°C hold
2. Genomic DNA purification (QIAGEN, Genomic-tip 100/G)
2.1 Equilibrate the Genomic-tip 100/G with 4 ml QBT Buffer
2.2 WGA gDNA (combine the contents of 3 tubes) 300 ml
QBT Buffer 9.7 ml / 10 ml
® mix and apply to 2 equilibrated Genomic-tip 100/G (5 ml/tip)
2.3 Wash the columns with 7.5 ml QC Buffer
2.4 = Repeat step 2.3
2.5 Elute the WGA gDNA with 5 ml of 50°C prewarmed QF Buffer
2.6 Combine the eluates of 2 columns and add 7 ml isopropanol, mix and centrifuge at > 5,000 g for >15 min at 4°C
2.7 Wash with 70% Ethanol and air-dry the pellets
2.8 Resuspend the WGA gDNA in 0.1 ml of Water
2.9 Adjust the DNA concentration to 100 mg/88 ml Water
3. Fragmentation
3.1 Mix
Purified WGA gDNA 85.5 ml
10X Fragmentation buffer (Affymetrix P/N 900422) 9.5 ml / 95 ml
3.2 Dilute the Fragmentation Reagent (Affymetrix P/N 900131) as follows.
Fragmentation Reagent (3U/ml) 2 ml
10X Fragmentation buffer 6 ml
Water 52 ml / 60 ml
3.3 Mix
gDNA at the step 3.1 95 ml
Fragmentation Reagent (0.1 U/ml) 5 ml / 100 ml total
® 37°C x 30 min
® 95°C x 10 min
® 4°C hold
3.4 Gel electrophoresis (4% NuSieve 3:1 agarose) for 4 ml of the fragmented product, which should be a smear with the majority of the intensity at 25 - 200 bp.
4. Labeling
4.1 Place the following mix into a 0.2 mL tube
Water 3 ml
5X TdT buffer (Affymetrix P/N 900425) 30 ml
GeneChip DNA Labeling Reagent (Affymetrix P/N900484) 8 ml
TdT (Affymetrix P/N900426; 30 U/mL) 13 ml / 54 ml
4.2 Add the fragmented DNA at the step 3.3 to the mix at the step 4.1
® 37°C x 5 hr
® 95°C x 10 min
® 4°C hold
(can be stored at -20°C)
5. Concentration with Microcon YM-3 (Millipore, Amicon)
5.1 Add the product at the step 4.2 to a column
® centrifuge 14,000 x g at RT, until all supernatant will flow down (~50 min)
5.2 Add 300 ml water to the column
® centrifuge 14,000 x g at RT, until the supernatant volume will be < 40 ml (~45 min)
5.3 reverse the column into a new collection tube
® centrifuge 1,000 x g for 5 min at RT
(can be stored at -20°C)
6. Target Hybridization
6.1 Prepare the followings into a 0.5 mL tube
2X Hybridization Buffer (see Affymetrix GeneChip Expression Analysis Technical Manual) 100 ml
Human Cot-1 (1 mg/ml; Invitrogen P/N 15279-011) 25 ml
50X Denhardt’s solution (Sigma P/N D2532) 10 ml
100% DMSO (Sigma P/N D5879) 20 ml
3 nM Oligo B2 (Affymetrix P/N 500702 B2) 4 ml / 159 ml
6.2 Add 41 ml of the concentrated DNA at the step 5.3 to the tube at the step 6.1
® mix and spin down
6.3 Heat the solution at 95°C, and immediately cool to 4°C
® Inject 200 ml of the denatured cocktail into one HGU133 Plus 2.0 array
6.4 Hybridize at 48°C for 16 hr at 60 rpm
7. Washing, Staining and Scanning
7.1 Prepare the following buffers.
(Pre-wash Buffer)
20X SSPE 1 ml
5M TMACl (Sigma P/N T3411) 25 ml
10% Tween-20 (Pierce P/N 28320) 0.05 ml
Water 23.95 ml / 50 ml
® Filtrate with a 0.2 mm filter
(Wash Buffer A & B) see Affymetrix GeneChip Mapping 100K Assay Manual
(2X Staining Buffer) see Affymetrix GeneChip Expression Analysis Technical Manual
(Streptavidin solution; first stain)
2X Staining Buffer 300 ml
Acetylated BSA (Invitrogen P/N 15561-020, 50 mg/ml) 24 ml
Streptavidin (1 mg/ml, Pierce P/N 21122) 6 ml
Water 270 ml / 600 ml
(Antibody stain solution; second stain)
2X Staining Buffer 300 ml
Acetylated BSA (50 mg/ml) 24 ml
Goat IgG (Sigma P/N I5256, 10 mg/ml) 6 ml
Biotinylated anti-streptavidin antibody (Vector P/N BA-0500, 0.5 mg/ml) 6 ml
Water 264 ml / 600 ml
(SAPE stain solution; third stain)
2X Staining Buffer 300 ml
Acetylated BSA (50 mg/ml) 24 ml
SAPE (Molecular Probes P/N S866, 1 mg/ml) 6 ml
Water 270 ml / 600 ml
7.2 Remove Hybridization cocktail from array and fill with 200 ml of Pre-wash Buffer
7.3 Incubate the array in Hybridization oven at 50°C for 30 min at 60 rpm
7.4 For Fluidics Station
Prime the lines with the Protocol; “PRIME”
Line A: Wash Buffer A (Non-Stringent Wash Buffer)
Line B: Wash Buffer B (Stringent Wash Buffer)
*Edit the Protocol; “FlexGE-WS2v4_450” as follows, and click “Run”
FlexGE-WS2v4_450Parameter values for modification
Post hyb wash #1 / 10 cycles of 5 mixes/cycle with Wash Buffer A at 35°C
Post hyb wash #2 / 40 cycles of 10 mixes/cycle with Wash Buffer B at 50°C
Stain / Stain the probe array for 10 minutes in Streptavidin solution at 35°C
Post stain wash / 10 cycles of 4 mixes/cycle with Wash Buffer A at 35°C
Second stain / Stain the probe array for 10 minutes in Antibody stain solution at 35°C
Third stain / Stain the probe array for 10 minutes in SAPE stain solution at 35°C
Final wash / 15 cycles of 4 mixes/cycle with Wash Buffer A at 35°C.
The holding temperature is 25°C.
7.5 Run “Scan” for scanning the arrays
Primers used for real-time PCR in Figure 3a
For the 1p34.2 region
Forward: 5'-GTGCTTTGACTGGATGTGTGAGAG-3'
Reverse: 5'-GCACTAAAAAGGCGGGAGTAAGAG-3'
For the 1q22 region
Forward: 5'-TCATTTGCTTGCTTAAAGCCTCTG-3'
Reverse: 5'-TGGGTATGGAAGGAGTATGTGTGG-3'
For the 1q32.1 region
Forward: 5'-ACAGACAATAGCTGCCTGGAGTTG-3'
Reverse: 5'-GACCCTGTAGTAGGCCACTGCTTT-3'
For the 9q31.3 region
Forward: 5'-TGAGGTTTGATGCACAGTAAAAGGA-3'
Reverse: 5'-GGCCAATTAGACAAAAACATTCAGG-3'
For the 9q33.1 region
Forward: 5'-GCTCCTCAGATTCAGGTCTCCTTC-3'
Reverse: 5'-GCCTATCTTTCAAAGGCTTCGTGT-3'
For the 8p23.2 region
Forward: 5'-ATATAGCCTGTGCAATGCCACCAA-3'
Reverse: 5'-GCTCATTATGAAGACTCCCATTCC-3'
For the8p23.1 region
Forward: 5'-TGTTGTGCAACCATCACCACTATC-3'
Reverse: 5'-AGGTCCCTAGAACAGCCAGATTCA-3'
For the 8p22 region
Forward: 5'-GACAGGTGTTATGCACTGGTGTTG-3'
Reverse: 5'-ATGCCAGAAAATGAAGGCTAACTG-3'
For the 8p21.1 region
Forward: 5'-AGGATTTTAGGGTTGGATGGGTTT-3'
Reverse: 5'-GGAATGACTGATATTGCCCTTGAC-3'
For GAPDH gene
Forward: 5'-CTGACCTGCCGTCTAGAAAAACCT-3'
Reverse: 5'-CAGGAAATGAGCTTGACAAAGTGG-3'
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