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 l
5M KOH
40 l
0.5M EDTA (pH 8.0)
10 l / 500 l
(Buffer D1) Water
35 l
Solution A
5 l / 40 l (for ≤5 samples)
(Buffer N1) Water
72 l
Solution B in the kit
8 l / 80 l (for ≤5 samples)
1.2
Water
2 l
Genomic DNA (50 ng/l in TE0.1)
0.5 l
Buffer D1
2.5 l / 5l
prepare 3 tubes per sample, mix and spin down,  RT x 3 min
1.3
add
Buffer N1
5 l to each tube
mix and spin down
1.4
prepare the following master mix (90 l per tube at the step 1.3) in a 0.2 ml tube on ice
Water
64 l
REPLI-g Buffer, 4x
25 l
REPLI-g DNA Polymerase
1 l / 90l
1.5
To the above tube, add 10 l 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 l
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 g/88 l Water
3. Fragmentation
3.1
Mix
Purified WGA gDNA
85.5 l
1
10X Fragmentation buffer (Affymetrix P/N 900422)
9.5 l / 95l
3.2
Dilute the Fragmentation Reagent (Affymetrix P/N 900131) as follows.
Fragmentation Reagent (3U/l)
2 l
10X Fragmentation buffer
6 l
Water
52 l / 60 l
3.3
Mix
gDNA at the step 3.1
95 l
Fragmentation Reagent (0.1 U/l)
5 l / 100 l 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 l 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 l
5X TdT buffer (Affymetrix P/N 900425)
30 l
GeneChip DNA Labeling Reagent (Affymetrix P/N900484) 8 l
TdT (Affymetrix P/N900426; 30 U/L)
13 l / 54l
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 l water to the column
centrifuge 14,000 x g at RT, until the supernatant volume will be < 40 l (~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 l
Human Cot-1 (1 mg/ml; Invitrogen P/N 15279-011)
25 l
50X Denhardt’s solution (Sigma P/N D2532)
10 l
100% DMSO (Sigma P/N D5879)
20 l
3 nM Oligo B2 (Affymetrix P/N 500702 B2)
4 l / 159l
2
Add 41 l 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
2
6.4
Inject 200 l of the denatured cocktail into one HGU133 Plus 2.0 array
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 m 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 l
Acetylated BSA (Invitrogen P/N 15561-020, 50 mg/ml)
24 l
Streptavidin (1 mg/ml, Pierce P/N 21122)
6 l
Water
270 l / 600 l
Antibody stain solution; second stain)
2X Staining Buffer
300 l
Acetylated BSA (50 mg/ml)
24 l
Goat IgG (Sigma P/N I5256, 10 mg/ml)
6 l
Biotinylated anti-streptavidin antibody (Vector P/N BA-0500, 0.5 mg/ml) 6 l
Water
264 l / 600 l
SAPE stain solution; third stain)
2X Staining Buffer
300 l
Acetylated BSA (50 mg/ml)
24 l
SAPE (Molecular Probes P/N S866, 1 mg/ml)
6 l
Water
270 l / 600 l

Remove Hybridization cocktail from array and fill with 200 l 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_450
Parameter 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
3
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'
4