femoral and humeral bones of balb / c mouse embryos were removed aseptically on 18.5  dpc , and they were microdissected into esz and growth plate ( egp ) under a stereomicroscope ( zeiss stemi 2000-c , carl zeiss microimaging ) .
each region was minced and gently digested with 2  mg / ml of collagenase ( wako pure chemical ) at 37  c for 2  h. they were cultured in growth medium composed of iscove 's modified dulbecco 's medium ( invitrogen ) supplemented with 15% fetal bovine serum , and subjected immediately to retroviral infection .
fli1 was introduced into the pmys - ires - gfp vector . the full length ews 
the per cell normalization method ( percellome method ) was applied to esz and egp samples .
a 10  l aliquot of each lysate was treated with dnase - free rnase a ( nippon gene inc . ,
japan ) for 30  min at 37  c , followed by proteinase k ( roche diagnostics gmbh .
usa ) was added to each well , shaken for 10  s four times and then incubated for 2  min at 30  c .
dna concentration was measured using a 96 well fluorescence plate reader with excitation at 485  nm and emission at 538  nm .
usa ) was used as standard . as reported previously , the grade - dosed spike cocktails ( gscs ) made of the bacillus subtilis rnas corresponding to the sequences in the affymetrix genechip arrays ( affx - thrx-3_at , affx - lysx-3_at , affx - phex-3_at , affx - dapx-3_at , and affx - trpnx-3_at ) were prepared , and gscs were added to the sample homogenates in proportion to their dna concentrations .
the genechip mouse genome 430 2.0 array ( affymetrix ) was hybridized with the crna generated from esz and egp cells , and murine ewing 's sarcoma tissue ( table  1 ) . after staining with streptavidin 
phycoerythrin conjugates , arrays were scanned using an affymetrix genechip scanner 3000 and analyzed using affymetrix genechip command console software ( agcc , affymetrix ) and genespring gx 11.0.2 ( agilent technologies ) as described previously .
the expression data for esz and egp cells were converted to percellome data , i.e. , absolute copy numbers of mrna per one cell , by the homemade software scal4 ( spike calculation version 4 ) .
response linearity of the five gsc spikes and the location of spike probe sets in the histogram of all probe sets ( fig . 
n  =  3 ) , all the pairs were plotted to a scatter graph as red ( expression above detection level ) or green dots ( below detection level ) with the data of five yellow spike probe sets ( fig . 
if any samples did not draw a symmetric scatter plot with yellow dot on the diagonal line , the sample were rejected for evaluation , and they were subjected to additional analyses .
homemade software named rsort ( roughness sort )   was used . this program sorts the probe sets as upward or downward peaks in a 3d isobologram ( fig .  2 ) . to avoid biologically nonsense
probe sets such as ones with expression below the detection level , the data were visually checked for their 3d isobologram shape .
femoral and humeral bones of balb / c mouse embryos were removed aseptically on 18.5  dpc , and they were microdissected into esz and growth plate ( egp ) under a stereomicroscope ( zeiss stemi 2000-c , carl zeiss microimaging ) .
each region was minced and gently digested with 2  mg / ml of collagenase ( wako pure chemical ) at 37  c for 2  h. they were cultured in growth medium composed of iscove 's modified dulbecco 's medium ( invitrogen ) supplemented with 15% fetal bovine serum , and subjected immediately to retroviral infection .
fli1 was introduced into the pmys - ires - gfp vector . the full length ews 
the per cell normalization method ( percellome method ) was applied to esz and egp samples . briefly
a 10  l aliquot of each lysate was treated with dnase - free rnase a ( nippon gene inc . ,
japan ) for 30  min at 37  c , followed by proteinase k ( roche diagnostics gmbh .
usa ) was added to each well , shaken for 10  s four times and then incubated for 2  min at 30  c .
dna concentration was measured using a 96 well fluorescence plate reader with excitation at 485  nm and emission at 538  nm .
usa ) was used as standard . as reported previously , the grade - dosed spike cocktails ( gscs ) made of the bacillus subtilis rnas corresponding to the sequences in the affymetrix genechip arrays ( affx - thrx-3_at , affx - lysx-3_at , affx - phex-3_at , affx - dapx-3_at , and affx - trpnx-3_at ) were prepared , and gscs were added to the sample homogenates in proportion to their dna concentrations .
the genechip mouse genome 430 2.0 array ( affymetrix ) was hybridized with the crna generated from esz and egp cells , and murine ewing 's sarcoma tissue ( table  1 ) . after staining with streptavidin 
phycoerythrin conjugates , arrays were scanned using an affymetrix genechip scanner 3000 and analyzed using affymetrix genechip command console software ( agcc , affymetrix ) and genespring gx 11.0.2 ( agilent technologies ) as described previously .
the expression data for esz and egp cells were converted to percellome data , i.e. , absolute copy numbers of mrna per one cell , by the homemade software scal4 ( spike calculation version 4 ) .
response linearity of the five gsc spikes and the location of spike probe sets in the histogram of all probe sets ( fig . 
n  =  3 ) , all the pairs were plotted to a scatter graph as red ( expression above detection level ) or green dots ( below detection level ) with the data of five yellow spike probe sets ( fig . 
if any samples did not draw a symmetric scatter plot with yellow dot on the diagonal line , the sample were rejected for evaluation , and they were subjected to additional analyses .
homemade software named rsort ( roughness sort )   was used . this program sorts the probe sets as upward or downward peaks in a 3d isobologram ( fig .  2 ) . to avoid biologically nonsense probe sets such as ones with expression below the detection level ,
we describe a unique dataset of mouse embryonic cartilage with or without the ewing 's sarcoma fusion oncogene , ews  fli1 .
the dataset was used in the study published recently   and was informative to understand the tumorigenic mechanisms of ewing 's sarcoma .
 ewing 's sarcoma is a rare bone tumor that affects children and adolescents . 
 we have recently succeeded to induce ewing 's sarcoma - like small round cell tumor in mice by expression of ews  ets fusion genes in murine embryonic osteochondrogenic progenitors . 
 the ewing 's sarcoma precursors are enriched in embryonic superficial zone ( esz ) cells of long bone . to get insights into the mechanisms of ewing 's sarcoma development , gene expression profiles between ews  
 fli1-sensitive esz cells and ews  
 fli1-resistant embryonic growth plate ( egp ) cells were compared using dna microarrays . 
 gene expression of esz and egp cells ( total , 30 samples ) was evaluated with or without ews  
 fli1 expression 0 , 8 or 48  h after gene transduction . 
 our data provide useful information for gene expression responses to fusion oncogenes in human sarcoma . 