SpecificationsOrganism/cell line/tissue*Mus musculus*Sexn.d.Sequencer or array typeNimbleGen 720K mouse whole genome tiling arrays.Data formatRaw data: PAIR file, analyzed data: txt fileExperimental factorsSFME cells vs normal mouse genomic DNA, SFME cells grown as spheres and after differentiation induction using TGF-ß or FCSExperimental featuresSFME cells were grown as spheres for undifferentiated state. Differentiation was induced by withdrawal of EGF and addition of TGF-ß or FCS. Array-CGH experiments were done with undifferentiated cells, 24 h-TGF-ß differentiation induced cells and 12 h-FCS differentiation induced cells.Consentn/aSample source locationSFME cells (CRL-9392™) from ATCC

1. Direct link to deposited data {#s0005}
================================

Deposited data can be found here: <http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE35523>.

2. Experimental design, materials and methods {#s0010}
=============================================

2.1. Cell culture and differentiation {#s0015}
-------------------------------------

SFME cells cultured in the absence of fibronectin formed spheres and served as non-differentiated controls. SFME cells were seeded on fibronectin-coated cultureware and allowed to grow for 18 h prior to differentiation induction with TGF-β or FCS. SFME cells were differentiation induced using above supplemented ATCC DMEM:F12 Medium containing TGF-β (10 ng/ml) for 8 h, 12 h and 24 h or DMEM:F12 supplemented with FCS for 8 h, 12 h and 24 h.

Cells were harvested and cell pellet was frozen before proceeding to DNA extraction as described previously (Fischer et al., 2014 genomics data) [@bb0005].

2.2. Array-CGH data analysis {#s0020}
----------------------------

Array data were deposited in GEO under accession number [GSE35523](ncbi-geo:GSE35523){#ir0015}.

Signal intensity data were extracted from scanned images of each array using Roche NimbleGen NimbleScan v2.6 software. After spatial correction, the Cy3 and Cy5 signal intensities were normalized using qspline normalization. Following normalization a 10 × window--averaging step is applied. For amplification and deletion detection we used the dynamic segMNT algorithm that identifies segments by minimizing the squared error relative to the segment means. To detect representative alterations and to minimize the identification of random alterations, we extracted segments with segment means greater than 0.1 threshold and a size greater than 250 kb. Deletions detected in undifferentiated, TGF-ß differentiation induced and FCS differentiation induced cells were summarized in [Table 1](#t0005){ref-type="table"}.

We used this low threshold of 0.1 for amplification and deletion detection because we were using a mixture of cells. Fluorescence in situ hybridization experiments at a single cell level had shown that gene amplifications were present at various percentages of the cells and in various copy numbers per single cell [@bb0010]. For further confirmation of the usefulness of a low threshold we did TaqMan copy number assay for two amplified genes namely *GFAP* and *FZR1*. *GFAP* revealed a 0.347 log~2~ ratio and *FZR1* a 0.2 log~2~ ratio.

2.3. qPCR analysis {#s0025}
------------------

TaqMan Copy Number Assays for genes *GFAP* and *FZR1* were performed following manufacturer\'s instructions. We used the *TERT* TaqMan Copy Number reference assay for relative quantitation of copy number of target genes. Mouse genomic DNA (Clontech) was used as control standard for normal diploid copy number. TaqMan assays were run in two independent experiments, each in four technical replicates and results were analyzed using StepOne™ Software v2.0 and copy numbers were analyzed using CopyCaller™ software. Mean results of four technical replicates were summarized in [Fig. 1](#f0005){ref-type="fig"}a (*GFAP*) and b (*FZR1*). The copy number calculated by Software Copy Caller™ revealed an increased copy number 3-fold of *GFAP* for SFME cell differentiation induced by TGFß for 8 h, 12 h and 24 h. In SFME cell differentiation induced by FCS for 8 h, 12 h and 24 h, the copy number was 2.5, 3 and 2.5-fold respectively. The software also identified an increased copy number of 2.5-fold for *FZR1* for SFME cell differentiation induced by TGFß for 8 h, 12 h and 24 h. Likewise we found an increased copy number of 2.5-fold for SFME cell differentiation induced by FCS for 24 h. These results confirmed our previous array-CGH analysis and FISH experiments. Interestingly the higher log~2~ ratio values for *GFAP* in array-CGH experiments corresponded to an elevated copy number value in TaqMan qPCR experiments.

3. Discussion {#s0030}
=============

Here we report detailed information on threshold choice for detection of gene amplification using NimbleGen 730K mouse whole genome array and correlation between log~2~ ratio values and copy number values from TaqMan qPCR experiments. Here and in our previous report we detected a complex pattern of amplifications and deletions. Both amplifications and deletions were only detectable after a low threshold setting. Threshold settings of 0.8 used in many studies were very likely to miss alterations that were present in a subpopulation of the investigated cells. Our confirmation using qPCR strongly argues for a low threshold setting. This dataset is an additional step towards uncovering copy number changes upon differentiation in mammalian stem cells.

The "Deutsche Forschungsgemeinschaft" funded this study (Fi644/2-1; Fi644/2-2).

![Amplification analysis using q-PCR.\
Amplification of *GFAP* and *FZR1* was analyzed by qPCR using the TaqMan copy number assays. SFME cells grown as spheres served as undifferentiated controls. SFME cells were investigated at three different time points with TGF-ß and FCS differentiation induction. Mouse genomic DNA served as standard for normal diploid copy number. The average copy number was 3 of *GFAP* in SFME cell differentiation induced by TGF-ß for 8 h, 12 h and 24 h. In SFME cell differentiation induced by FCS for 8 h, 12 h and 24 h, the average copy number was 2.5, 3 and 2.5 respectively. The average copy number was 2.5 for *FZR1* in SFME cell differentiation induced by TGF-ß for 8 h, 12 h and 24 h, induced by FCS for 24 h. There was no copy number gain for *FZR1* detectable in SFME cell differentiation induced by FCS for 8 h and 12 h.](gr1){#f0005}

###### 

Overview of deleted chromosome regions.

Start and end points of deleted chromosome regions are according to NCBI37/mm9. Size is displayed in kb.

  Sphere   24 h TGF-ß   12 h FCS                                                                                                                          
  -------- ------------ ----------- ----------- -------- ------- ----------- ----------- ----------- -------- ------- ----------- ----------- ----------- --------
                                                         chr1    3019999     9259999     − 0.11308   6240                                                 
                                                         chr1    10419999    12539999    − 0.16881   2120                                                 
                                                         chr1    21459999    33339999    − 0.12453   11,880                                               
                                                         chr1    47059999    51179999    − 0.20953   4120                                                 
                                                         chr1    67859999    68979999    − 0.20877   1120                                                 
                                                         chr1    95859999    106179999   − 0.11556   10,320                                               
                                                         chr1    108899999   120099999   − 0.13136   11,200                                               
  chr1     110459999    112459999   − 0.15437   2000                                                                                                      
  chr1     125099999    125779999   − 0.1133    680                                                                                                       
                                                         chr1    141739999   151499999   − 0.12807   9760                                                 
  chr1     157499999    166019999   − 0.11672   8520                                                          chr1    157699999   164339999   − 0.11251   6640
                                                                                                              chr1    179699999   180099999   − 0.17032   400
                                                         chr2    39299999    49379999    − 0.11759   10,080                                               
                                                         chr2    80899999    83139999    − 0.16313   2240                                                 
  chr2     85619999     89979999    − 0.1151    4360     chr2    85539999    89979999    − 0.19832   4440                                                 
  chr2     94819999     101179999   − 0.10144   6360     chr2    94419999    101179999   − 0.14893   6760                                                 
  chr2     140259999    140739999   − 0.10916   480                                                                                                       
  chr2     174619999    176979999   − 0.10819   2360     chr2    174539999   176979999   − 0.10409   2440                                                 
                                                         chr3    3179999     7819999     − 0.16459   4640                                                 
  chr3     10779999     15219999    − 0.10217   4440     chr3    10699999    14179999    − 0.21013   3480                                                 
  chr3     15259999     15819999    − 0.25966   560      chr3    15339999    18379999    − 0.18784   3040     chr3    15459999    15779999    − 0.2969    320
                                                         chr3    23219999    26019999    − 0.12403   2800                                                 
                                                         chr3    41659999    48699999    − 0.20394   7040                                                 
  chr3     47419999     48019999    − 0.20803   600      chr3    48739999    50819999    − 0.10307   2080                                                 
                                                         chr3    66659999    67219999    − 0.14445   560                                                  
                                                         chr3    69859999    71299999    − 0.14      1440                                                 
  chr3     71339999     73539999    − 0.1502    2200     chr3    71339999    72859999    − 0.24188   1520                                                 
                                                         chr3    72899999    75019999    − 0.12964   2120                                                 
                                                         chr3    76179999    78579999    − 0.10363   2400                                                 
  chr3     80059999     81099999    − 0.14237   1040     chr3    80659999    81059999    − 0.20763   400                                                  
  chr3     93699999     94059999    − 0.18368   360      chr3    93699999    94059999    − 0.16031   360                                                  
                                                         chr3    110219999   115099999   − 0.16927   4880                                                 
                                                         chr3    116859999   120779999   − 0.1109    3920                                                 
  chr3     123499999    125779999   − 0.2668    2280     chr3    123339999   125779999   − 0.35956   2440     chr3    123059999   127819999   − 0.16916   4760
  chr3     125819999    127739999   − 0.16144   1920     chr3    125819999   128939999   − 0.16294   3120                                                 
                                                         chr3    131419999   132379999   − 0.13963   960                                                  
                                                         chr3    140139999   140899999   − 0.18969   760                                                  
                                                         chr3    149859999   151299999   − 0.18356   1440                                                 
                                                         chr3    154499999   159578619   − 0.10621   5079                                                 
                                                         chr4    12379999    32099999    − 0.13785   19,720                                               
                                                         chr4    35779999    39859999    − 0.20166   4080                                                 
                                                         chr4    64579999    75579999    − 0.11545   11,000                                               
  chr4     75659999     80779999    − 0.2069    5120     chr4    75619999    80779999    − 0.2917    5160     chr4    75579999    78339999    − 0.19677   2760
                                                         chr4    89259999    94339999    − 0.13013   5080                                                 
                                                         chr5    5939999     7859999     − 0.15729   1920                                                 
                                                         chr5    11859999    19539999    − 0.12821   7680                                                 
                                                         chr5    54859999    61859999    − 0.20136   7000                                                 
                                                         chr5    67699999    72699999    − 0.13854   5000                                                 
                                                         chr5    78299999    91059999    − 0.10839   12,760   chr5    81459999    81739999    − 0.14919   280
                                                                                                              chr5    146259999   146579999   − 0.10129   320
  chr6     41499999     47339999    − 0.10686   5840     chr6    41539999    47099999    − 0.16256   5560                                                 
                                                         chr6    55739999    66419999    − 0.10699   10,680                                               
                                                         chr6    73379999    81459999    − 0.11413   8080                                                 
                                                         chr6    103779999   112019999   − 0.1065    8240                                                 
  chr6     138299999    140059999   − 0.14334   1760     chr6    138379999   140059999   − 0.22473   1680                                                 
                                                         chr7    56899999    70659999    − 0.10699   13,760                                               
                                                         chr7    75539999    79379999    − 0.11891   3840                                                 
                                                         chr7    91779999    103339999   − 0.10018   11,560                                               
  chr7     10699999     12139999    − 0.24888   1440                                                                                                      
  chr7     110659999    111699999   − 0.23809   1040     chr7    110619999   111379999   − 0.20134   760                                                  
                                                         chr8    4579999     9659999     − 0.11752   5080                                                 
  chr8     99539999     106019999   − 0.10809   6480                                                                                                      
                                                         chr8    29979999    34539999    − 0.13338   4560                                                 
                                                         chr8    49459999    55619999    − 0.16084   6160                                                 
                                                         chr8    98739999    106419999   − 0.17182   7680                                                 
                                                         chr9    3139999     7299999     − 0.10507   4160                                                 
                                                         chr9    10419999    12939999    − 0.15382   2520                                                 
                                                         chr9    16739999    20259999    − 0.10813   3520                                                 
                                                         chr9    33219999    34019999    − 0.11795   800                                                  
  chr9     35659999     35939999    − 0.21324   280      chr9    35659999    36299999    − 0.16454   640      chr9    35659999    36059999    − 0.16676   400
  chr9     37699999     38899999    − 0.10023   1200     chr9    37419999    39979999    − 0.10149   2560                                                 
                                                                                                              chr9    71699999    72019999    − 0.1087    320
                                                         chr9    115059999   115619999   − 0.10853   560                                                  
                                                         chr10   15059999    16899999    − 0.1919    1840                                                 
  chr10    35579999     35939999    − 0.17695   360      chr10   26739999    38059999    − 0.11928   11,320                                               
  chr10    45899999     51019999    − 0.12617   5120     chr10   45899999    49499999    − 0.20934   3600     chr10   47739999    49099999    − 0.13582   1360
                                                         chr10   63059999    65899999    − 0.11266   2840                                                 
                                                         chr10   71099999    74299999    − 0.10106   3200                                                 
  chr10    100819999    105099999   − 0.16547   4280     chr10   100819999   105099999   − 0.22175   4280     chr10   101059999   104739999   − 0.11145   3680
                                                         chr10   111619999   114339999   − 0.19567   2720                                                 
                                                         chr10   122579999   126139999   − 0.11643   3560                                                 
  chr10    128539999    129975647   − 0.10464   1436     chr10   128539999   129975647   − 0.14084   1436                                                 
                                                         chr11   8979999     18619999    − 0.11164   9640     chr11   17579999    18619999    − 0.10657   1040
  chr11    36019999     42459999    − 0.11313   6440     chr11   36459999    42459999    − 0.19152   6000                                                 
                                                         chr11   90499999    93299999    − 0.14477   2800                                                 
                                                         chr12   89939999    99619999    − 0.12512   9680                                                 
                                                         chr12   114979999   116219999   − 0.10697   1240                                                 
                                                         chr13   76539999    90659999    − 0.12234   14,120                                               
                                                         chr13   115939999   120282113   − 0.12049   4342                                                 
                                                         chr14   49819999    53379999    − 0.12933   3560     chr14   50499999    52099999    − 0.12137   1600
                                                                                                              chr14   52379999    53419999    − 0.10299   1040
  chr14    76859999     78259999    − 0.10653   1400                                                          chr14   76939999    125175837   − 0.15227   48,236
  chr14    88619999     95699999    − 0.17732   7080     chr14   80339999    98619999    − 0.31002   18,280                                               
                                                         chr14   98659999    106899999   − 0.14224   8240                                                 
                                                         chr14   106939999   118059999   − 0.26608   11,120                                               
                                                         chr14   118099999   125175837   − 0.13545   7076                                                 
                                                         chr15   9419999     10299999    − 0.13815   880                                                  
  chr15    13459999     23819999    − 0.1377    10,360   chr15   13339999    24459999    − 0.20317   11,120                                               
                                                                                                     6360     chr15   13499999    14979999    − 0.10807   1480
  chr15    46139999     47259999    − 0.15127   1120     chr15   44659999    51019999    − 0.17027            chr15   19699999    22619999    − 0.10222   2920
  chr15    47419999     51059999    − 0.12808   3640                                                                                                      
                                                         chr15   89459999    90219999    − 0.12766   760                                                  
                                                         chr16   59059999    89699999    − 0.13622   30,640                                               
  chr17    17499999     22579999    − 0.11022   5080     chr17   18299999    20899999    − 0.2772    2600                                                 
                                                         chr17   37259999    42659999    − 0.17895   5400                                                 
                                                         chr17   50899999    56019999    − 0.11015   5120                                                 
                                                         chr17   57539999    62859999    − 0.14589   5320                                                 
  chr17    76139999     78259999    − 0.13494   2120     chr17   76139999    78299999    − 0.17557   2160                                                 
                                                         chr17   81499999    83219999    − 0.10341   1720                                                 
  chr17    89499999     95255954    − 0.10669   5756     chr17   89259999    95255954    − 0.15303   5996                                                 
  chr18    16939999     19899999    − 0.11972   2960     chr18   16979999    19779999    − 0.1813    2800     chr18   7899999     8339999     − 0.13658   440
  chr18    26139999     31459999    − 0.11968   5320     chr18   26219999    31379999    − 0.21126   5160                                                 
  chr18    51059999     52299999    − 0.14649   1240     chr18   50739999    52659999    − 0.19077   1920     chr18   51659999    52099999    − 0.12795   440
                                                         chr18   70659999    73179999    − 0.1304    2520                                                 
  chr18    75979999     76259999    − 0.16726   280                                                           chr18   75779999    76499999    − 0.11312   720
  chr18    85659999     90459999    − 0.1178    4800     chr18   85299999    90765552    − 0.13801   5466                                                 
                                                         chr19   47779999    52739999    − 0.12561   4960                                                 
