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Supplementary Methods
Selection criteria for common insertion sites (CISs)
TAPDANCE software was used to identify CIS and carry out association analyses
(manuscript in press). Starting with barcoded sequence data, the software identifies and
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trims sequences and maps putative genomic sequence to a reference genome using the
bowtie short read mapper. Poisson distribution statistics are then applied to assess and
rank genomic regions showing significant enrichment for transposon insertion. Novel
methods of counting insertions are used to ensure that the results presented have the
expected characteristics of informative CISs. A persistent mySQL databases is generated
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and utilized to keep track of sequences, mappings and common insertion sites.
Additionally, associations between phenotypes and CISs are also identified using Fisher’s
exact test with multiple testing correction.
RT-PCR
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Extraction of RNA from tumor nodules was done using the Trizol reagent (Invitrogen)
using protocols described by the manufacturer. First strand cDNA synthesis was
performed using the Transcriptor First Strand cDNA Synthesis Kit (Roche) as described
by the manufacturer using 1 µg total RNA as template. Both reactions using with (RT+)
and without (RT–) the reverse transcriptase were performed for all the samples.
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Subsequent PCR was performed using 1 µl of the cDNA as template with various primer
pairs. Primer sequences for alpha-fetoprotein (Afp) were forward 5’CCTGTGAACTCTGGTATCAG-3’ and reverse 5’-GCTCACACCAAAGCGTCAAC-3’
(amplicon 410 bp); secreted phosphoprotein 1 (Spp1) forward 5’CTTTCACTCCAATCGTCCCTAC-3’ and reverse 5’-
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GCTCTCTTTGGAATGCTCAAGT-3’ (amplicon 305 bp); actin beta (Actb) forward 5’GTGACGAGGCCCAGAGCAAGAG-3’ and reverse 5’AGGGGCCGGACTCATCGTACTC-3’ (amplicon 938 bp); fumarylacetoacetate
hydrolase (Fah) forward 5’-ATGAGCTTTATTCCAGTGGCC-3’ and reverse 5’ACCACAATGGAGGAAGCTCG-3’ (amplicon 503 bp); truncated epidermal growth
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factor receptor (EGFR) forward 5’-GACCCCCAGCGCTACCTTGTCATTCAG-3’ and
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reverse (specific for the rabbit -globin polyA) 5’GCCACACCAGCCACCACCTTCTG-3’ (amplicon 140 bp); green fluorescent protein
(Gfp) forward 5’-ACTTGTACAGCTCGTCCATGC-3’ and reverse 5’TCGTGACCACCCTGACCTAC-3’ (amplicon 537 bp); activated catenin beta 1
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(CTNNB1-S33Y) forward 5’-GCCGCTTACTTGTCATCGTCGTCCT-3’ and reverse 5’TGTTCCGAATGTCTGAGGACAAGCC-3’ (amplicon 400 bp); nuclear factor I/B
(Nfib) forward 5’-ATGATGTATTCTCCCATCTGTCTCACT-3’ and reverse 5’ATGTGGTGTGGCTGAACACAAAGTG-3’ (amplicon 506 bp); partitioning-defective
protein 3 (Pard3) forward 5’-GGAGATGGCCGCATGAAAGTTTTCA-3’ and reverse
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5’-TTCTGCTTGAGAAAGCCAGCTGTCG-3’ (amplicon 500 bp). PCR conditions are
similar to PCR genotyping described previously except 25 to 30 cycles were performed
to avoid amplicon saturation. Semi-quantitative analyses of non-saturated RT-PCR
amplicons were performed using the ImageJ 1.40J software (developed by NIH,
Maryland, USA). Briefly, intensity of non-saturated RT-PCR amplicon bands was
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measured as an arbitrary value relative to Actb expression levels.
Gene expression and copy number changes in human HCC samples
An initial 91 hepatitis C (HCV) related hepatocellular carcinoma samples were analyzed
for gene expression and DNA copy number changes. DNA and RNA extraction from
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human tissue, SNP-array technology (StyI chip of the 250K Human Mapping Array set
from Affymetrix), and gene expression microarray methodology (U133 Human Chip Plus
2.0 from Affymetrix) are extensively described elsewhere (1, 2). Real expression values
of EGFR for the training and validation sets as shown in Supplementary Data 4 and
Supplementary Data 5, respectively. Comparisons were conducted using the unpaired
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t-test and additional data analysis was conducted using the GenePattern Analytical
Toolkit (3), the R (http://www.R-project.org) and SPSS software® (version 18). A
second independent dataset including 144 HCV-related HCC formalin-fixed paraffinembedded (FFPE) tissue sections from various etiologies was analyzed for gene
expression (DASL, Illumina) (4). Finally, cytogenetic abnormalities were evaluated
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using fluorescent in situ hybridization (FISH) in 51 out of 144 samples, performed
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according to standard protocol. Briefly, commercial a-satellite probe corresponding to
the centromeric regions of chromosome 7 (CEP-7) was purchased prelabeled with
SpectrumAqua (Abbott Molecular/Vysis, Inc.). Four micron slides sections from
formalin-fixed HCC tissue blocks were baked at 60°C for at least two hours. Slides were
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soaked for 10 min in xylene, followed by 100% ethanol and 96% ethanol washes. The
slides were boiled in MES (2-[n-morpholino]ethanesulphonic acid) buffer for 10 min, and
washed twice in Tris-HCl buffer for 3 min. Slides were placed on a Thermobrite (IRIS
Sample Processing) held at 37°C to allow tissue digestion with Digest-All III (pepsin)
solution (Invitrogen) for 4 min. Samples were washed in Tris-HCl buffer for 2 min.
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Slides were dehydrated for 2 min each in the ethanol series 70%, 80%, 90% and 100%.
Probe was added to slides, which were then coverslipped and sealed with rubber cement.
Samples and probes were co-denatured on a ThermoBrite (IRIS Sample Processing) at
94°C for 5 min and hybridized overnight at 37°C on the ThermoBrite. Slides were
washed in Tris-HCl buffer for 2 min and then incubated at 70°C for 10 min in saline-
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sodium citrate (SSC), rinsed in room temperature, dehydrated for 2 min each in the
ethanol series 70%, 80%, 90%, 100%, and counterstained with DAPI (4',6-diamidino-2phenylindole, Abbott). Results of the hybridization were imaged using a Nikon Eclipse
50I fluorescence microscope. Individual images were captured using an Applied Imaging
system running CytoVision Genus (version 3.9). Signal counts were obtained from 100
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hundred nuclei per slide. Mean intensity of probes signal and probe ratio was calculated
for each analyzed sample. Polysomy was defined if mean probe signal was >2 in at least
40% of counted cells.
Hydrodynamic injection
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Hydrodynamic injections were performed as previously described (5-7). Briefly,
fumarylacetoacetate hydrolase (Fah)-null mice carrying the Rosa26-SB11 transgene
(Fah/SB11) were generated. Generation of pT2/PGK-FAHIL, a plasmid containing the
mouse Fah cDNA fused to the Luciferase reporter gene cassette flanked by SB
transposon inverted repeat/direct repeats (IR/DRs), has been previously described (7).
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The plasmid pT2/PGK-rabbit ß-globin polyA was first constructed using standard PCR
amplification for the human phosphoglycerate kinase (PGK) promoter and restriction
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enzyme digest of the rabbit ß-globin polyA from pCX-EGFP (a generous gift from Dr
Masaru Okabe, Osaka University). These two components were ligated into pT2/BH
(Largaespada laboratory, University of Minnesota) to obtain pT2/PGK-pA. Truncated
and full-length EGFR (a generous gift from Dr Lee Opresko, Pacific Northwest National
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Laboratory) were PCR amplified and ligated between the PGK promoter and rabbit ßglobin polyA to get pT2/PGK-Trunc. EGFR and pT2/PGK-EGFR, respectively, as
previously described. To generate an empty gene delivery plasmid (pKT2/GD-Empty)
for control injections and for constructing pKT2/GD-GFP, pKT2/Floxed-FAHIL was first
digested with EcoRI, blunted with Klenow fragment and self-ligated to obtain a
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pKT2/Floxed FAHIL vector lacking any EcoRI sites (pKT2/Floxed FAHIL-EcoRI-).
PGK-rabbit ß-globin polyA component from pT2/PGK-rabbit ß-globin polyA was
isolated by restriction enzyme digest, blunted and ligated to pKT2/Floxed-FAHIL-EcoRIto obtain the pKT2/GD-Empty vector. Gfp gene component was extracted from pCXEGFP using restriction enzyme digest and ligated into pKT2/GD-Empty to get
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pKT2/GD-Gfp. The Fah cDNA was also removed from pKT2/Floxed FAHIL by
restriction enzyme digest and ligated into pT2/PGK-pA to get pT2/PGK-Fah. An IRESGfp fluorescent component was introduced into pT2/PGK-Fah to get pT2/PGK-FahIRES-Gfp. To generate pKT2/GD-DEST plasmid, the destination cassette (DEST) from
pcDNA-DEST40 (Invitrogen) was removed by restriction enzymes and cloned into
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pKT2/GD-Empty to get pKT2/GD-DEST. The Fah cDNA was removed from
pKT2/GD-DEST by restriction enzymes to get pKT2/GD-DEST-Fah-. The internal
components of pKT2/GD-DEST-Fah- lacking the transposon IR/DRs were removed by
restriction enzyme digest and cloned into pT2/BH vector containing the rabbit ß-globin
polyA signal to obtain the final pT2/GD-DEST-Fah- vector. The Fah-IRES-Gfp
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component from pT2/PGK-Fah-IRES-Gfp was removed by restriction enzymes and
ligated into pT2/GD-DEST-Fah- to get the final pT2/GD-IRES-Gfp vector for clonase
reaction (Invitrogen). CTNNB1S33Y component from pcDNA-S33Y Beta-catenin
(Addgene) was cloned into pENTR entry vector (Invitrgen) to get pENTR-CTNNB1.
CTNNB1S33Y component from pENTR-CTNNB1 was cloned into pT2/GD-IRES-Gfp
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using clonase reaction to get pT2/GD-IRES-Gfp-CTNNB1. Generation of transposon
vector containing the short hairpin against Trp53 gene (pT2/shp53) has been previously
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described (5-7). All transgenes used for hydrodynamic tail vein injections were prepared
using Nucleobond AX plasmid DNA Maxi-preparation kits (Macherey-Nagel). Twenty
micrograms of each construct was hydrodynamically injected into 6-week old Fah/SB11
mice using previously established conditions (8). These mice were normally maintained
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with 7.5 µg/ml 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC)
drinking-water but replaced with normal drinking water after hydrodynamic injection of
transposon vectors. These experimental animals were observed for weight changes and
Luciferase activity as previously described (5-7).
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oPOSSUM analyses
oPOSSUM analysis (version 3) was performed using the mouse single site analysis
(http://opossum.cisreg.ca/cgi-bin/oPOSSUM3/opossum_mouse_ssa) for the detection of
over-represented transcription factor binding sites in the promoters of our CIS genes (9,
10). The search parameters used were: conservation cutoff, 0.4; matrix score threshold,
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80%; amount of upstream/downstream sequence, 5000/5000; number of results to return,
Z-score ≥ 10 and Fisher score ≥ 7.
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