nhnp cells ( p1 ) were grown in 75  cm cell culture flasks with npmm ( neuronal progenitor maintenance medium ) for initial 24  h after thawing . for the undifferentiated approach nhnp cells ( approximately 4    10 cells ) were cultivated for additional 24  h in npmm , harvested and the cell pellet was frozen before dna extraction . for differentiation induction nhnp cells were transferred to 25  cm laminin - coated cell culture flasks in npdm ( neural progenitor differentiation medium ) supplemented with bdnf ( brain - derived neurotrophic factor ) at 25  ng / ml . we used approximately 4    10 cells for 24  h differentiation induction , approximately 2.5    10 cells for 48  h differentiation induction and approximately 2.5    10 cells for 5d differentiation induction approach . cell pellets were resuspended in lysis buffer ( 75  mm nacl , 25  mm edta , ph  8) with 10% sds . undifferentiated nhnp cells , 24  h differentiation - induced and 5d differentiation - induced nhnp cell pellets were treated with proteinase k for >  18  h at 55  c . 48  h differentiation - induced nhnp cell pellets were treated with proteinase k for 5  h at 55  c . all samples were extracted with 6  m nacl / chloroform for 1  h on a rotator , centrifuged and the aqueous layer was precipitated with isopropanol and/or with sodium acetate ethanol . genomic dna from blood lymphocytes was extracted accordingly with proteinase k digest for >  18  h at 55  c . the array - cgh experiments were done with independently derived primary cells with different lot numbers . 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 0.1 threshold and a size greater than 250  kb . deletions detected in undifferentiated , 24  h differentiated , 48  h differentiated and 5 d - differentiated nhnp cells were summarized in table  1 . the array plots at 25  kb resolution obtained by segmentation algorithm impressively demonstrate changes of the complex pattern of different copy numbers along a given chromosome . 1 summarizes the array plots for all probes of chromosome 12 and fig .  2 of the array plots of all probes for chromosome 17 . however , only after a 1  day - differentiation a wavy pattern appears that increases in number and amplitude heights over time . recently , several studies explained the wavy cgh pattern by dna extraction and replication timing , . our results , however , do not support this hypothesis as we detected wavy cgh pattern indicative of imbalances in cells seeded for differentiation in different cell densities . in addition , dna digestion with proteinase k for 5  h or >  18  h did not lead to reduction of the wavy cgh pattern . in fact after 5d of differentiation and after more than 18  h protein digest we detected the highest amplitudes for copy number changes as shown in fig .  1 , fig .  2 . nhnp cells ( p1 ) were grown in 75  cm cell culture flasks with npmm ( neuronal progenitor maintenance medium ) for initial 24  h after thawing . for the undifferentiated approach nhnp cells ( approximately 4    10 cells ) were cultivated for additional 24  h in npmm , harvested and the cell pellet was frozen before dna extraction . for differentiation induction nhnp cells were transferred to 25  cm laminin - coated cell culture flasks in npdm ( neural progenitor differentiation medium ) supplemented with bdnf ( brain - derived neurotrophic factor ) at 25  ng / ml . we used approximately 4    10 cells for 24  h differentiation induction , approximately 2.5    10 cells for 48  h differentiation induction and approximately 2.5    10 cells for 5d differentiation induction approach . cell pellets were resuspended in lysis buffer ( 75  mm nacl , 25  mm edta , ph  8) with 10% sds . undifferentiated nhnp cells , 24  h differentiation - induced and 5d differentiation - induced nhnp cell pellets were treated with proteinase k for >  18  h at 55  c . 48  h differentiation - induced nhnp cell pellets were treated with proteinase k for 5  h at 55  c . all samples were extracted with 6  m nacl / chloroform for 1  h on a rotator , centrifuged and the aqueous layer was precipitated with isopropanol and/or with sodium acetate ethanol . genomic dna from blood lymphocytes was extracted accordingly with proteinase k digest for >  18  h at 55  c . the array - cgh experiments were done with independently derived primary cells with different lot numbers . 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 0.1 threshold and a size greater than 250  kb . deletions detected in undifferentiated , 24  h differentiated , 48  h differentiated and 5 d - differentiated nhnp cells were summarized in table  1 . the array plots at 25  kb resolution obtained by segmentation algorithm impressively demonstrate changes of the complex pattern of different copy numbers along a given chromosome . 1 summarizes the array plots for all probes of chromosome 12 and fig .  2 of the array plots of all probes for chromosome 17 . however , only after a 1  day - differentiation a wavy pattern appears that increases in number and amplitude heights over time . recently , several studies explained the wavy cgh pattern by dna extraction and replication timing , . our results , however , do not support this hypothesis as we detected wavy cgh pattern indicative of imbalances in cells seeded for differentiation in different cell densities . in addition , dna digestion with proteinase k for 5  h or >  18  h did not lead to reduction of the wavy cgh pattern . in fact after 5d of differentiation and after more than 18  h protein digest we detected the highest amplitudes for copy number changes as shown in fig .  1 , fig .  2 . here , we report detailed information on dna extraction method used for detection of copy number changes using nimblegen 730k whole genome array . here and in our previous report this wavy pattern of copy number changes was independent from cell number and protein digest duration . this dataset is a first step towards uncovering copy number changes upon differentiation in human stem cells .
dna sequence amplification occurs at defined stages during normal development in amphibians and flies and seems to be restricted in humans to drug - resistant and tumor cells only . 
 we used array - cgh to discover copy number changes including gene amplifications and deletions during differentiation of human neural progenitor cells . here , we describe cell culture features , dna extraction , and comparative genomic hybridization ( cgh ) analysis tailored towards the identification of genomic copy number changes . 
 further detailed analysis of amplified chromosome regions associated with this experiment , was published by fischer and colleagues in plos one in 2012 ( fischer et al . , 2012 ) . 
 we provide detailed information on deleted chromosome regions during differentiation and give an overview on copy number changes during differentiation induction for two representative chromosome regions .