although the msmo1, idi1 and hmgcs1 genes were all up - regulated in 293 - f and 293 - h compared to hek293, the down - regulation of the lathosterol oxidase gene ( sc5d ), which gene product is in downstream steps of the pathway, resulted in a predicted reduction in cholesterol production in 293 - f and 293 - h cells compared to hek293 ( supplementary fig. s7 ). comparisons between suspension cell lines ( 293 - f and 293 - h ) and adherent progeny cell lines ( 293e and 293t ) did not result in any predicted changes in cholesterol biosynthesis ( data not shown ). figure 5evaluation of common de genes between adherent and suspension hek293 cells identified cholesterol biosynthesis as the main enriched pathway. ( heat map with tpm values for each de gene common to all adherent to suspension comparisons. ( the top ten most enriched biological go terms of the 38 common de genes between adherent and suspension cells based on gene enrichment analysis. length and color of bars both show significance of adjusted p - value for the hypergeometric test. also, genes mentioned in each bar are the genes that belong to enriched go term and present in the list of 38 de genes. evaluation of common de genes between adherent and suspension hek293 cells identified cholesterol biosynthesis as the main enriched pathway. ( heat map with tpm values for each de gene common to all adherent to suspension comparisons. ( the top ten most enriched biological go terms of the 38 common de genes between adherent and suspension cells based on gene enrichment analysis. length and color of bars both show significance of adjusted p - value for the hypergeometric test. also, genes mentioned in each bar are the genes that belong to enriched go term and present in the list of 38 de genes. as four out of the 38 differentially expressed genes ( lox, smad7, id1 & txnip ) have previously been shown to have a role in the epithelial to mesenchymal transition ( emt ) pathway, we evaluated the role of this pathway in the transition from adherent to suspension cell growth of these hek293 cell lines. the normalized expression of a set of emt markers showed that the parental hek293 actually had the highest level of expression of various mesenchymal markers ( n - cadherin, vimentin and fibronectin ) of all the six cell lines ( supplementary fig. s8 ). moreover, when predicting emt pathway outcomes for suspension cells ( 293f and 293h ) compared to the parental hek293 strain using ipa, the results predicted reduced emt in the suspension progeny cells compared to hek293 ( supplementary fig. s8 ). however, suspension cells were predicted to have increased disrup tion of adherence junctions, which is consistent with the suspension cell phenotype. taken together the comparison between adherent and suspension hek293 progeny cell lines suggest that the transition between adherent to suspension cell growth is not equivalent to the epithelial to mesenchymal transition even though several emt - associated genes may be key to the difference between cell lines. instead key changes were found associated with cholesterol biosynthesis and fatty acid metabolism. for identification of key genes involved in the transition from adherent to suspension morphology, expression data from an additional set of 63 different human cell lines deposited in the human protein atlas database were analyzed. principal component analysis of these cell lines resulted in clustering of suspension cell lines in a distinc t group separated from adherent cell lines ( fig. 6a ). however, since most of the suspension cell lines are lymphoid or myeloid - derived this clustering may be a result of the similar origin of the suspension cell lines. transcription data of the 38 previously identified differentially expressed genes from 47 adherent and 16 suspension cell lines ( supplementary table s9 ) was compared between the two groups using a mann – whitney u - test. within this set, nine genes ( lox, id1, adamts1, zic1, kcnma1, dhrs3, rarg, col4a6 and arrdc4 ) had significant different expression levels between adherent and suspension cell lines with p - values < 0. 01 ( fig. 6b, c ). four of these genes ( adamts1, kcnma1, col4a6 and arrdc4 ) had the opposite directional change in the extended data set compared to the differential expression between only hek293 strains. based on these findings, the remaining five genes ( lox, id1, zic1, dhrs3 and rarg ), which showed a consistent down - regulation in suspension cell lines compared to adherent cells, may play important roles in the morphological differences between the adherent and suspension cell lines. figure 6gene expression validation of the 38 previously identified differentially expressed genes in 63 human cell lines, identified nine significantly differentially expressed genes between suspension and adherent cell lines. ( pca transcriptomic data of 63 human cell - lines from the human protein atlas shows a clear separation of suspension and adherent cell lines from different tissues. ( range of normalized counts in hpa cell lines for each of the previously identified 38 genes, differentially expressed between all adherent and suspension hek293 cell lines. the black line in each box shows median of normalized counts for the gene. ( genes that are differentially expressed between adherent and suspension cells using a mann – whitney u - test, with p - values < 0. 01, are highlighted in purple, where length of bars shows logarithmic fold change of median between two groups and the color of bars denotes degree of significance of p - value. non - significant genes have gray bars. gene expression validation of the 38 previously identified differentially expressed genes in 63 human cell lines, identified nine significantly differentially expressed genes between suspension and adherent cell lines. ( pca transcriptomic data of 63 human cell - lines from the human protein atlas shows a clear separation of suspension and adherent cell lines from different tissues. ( range of normalized counts in hpa cell lines for each of the previously identified 38 genes, differentially expressed between all adherent and suspension hek293 cell lines. the black line in each box shows median of normalized counts for the gene. ( genes that are differentially expressed between adherent and suspension cells using a mann – whitney u - test, with p - values < 0. 01, are highlighted in purple, where length of bars shows logarithmic fold change of median between two groups and the color of bars denotes degree of significance of p - value. non - significant genes have gray bars. due to the extensive usage of hek293 cells as a bioproduction platform for pharmaceutical proteins and aav vectors, characterization of the hek293 genome and transcriptome is relevant for bioprocess development. a deeper knowledge of the hek293 genomic and transcriptomic traits can for instance pave the way for more rational cell line engineering approaches, aiming to improve bioproduction efficiency and quality of protein products. as different hek293 lineages are propagated under different conditions and the observation that immortalized continuously cultured cell lines, such as hek293, have a high degree of genomic instability with frequent chromosome rearrangements it can be expected that different hek293 lineages are differentiated at the genomic and transcriptomic level compared to the parental cell line. here, the genomes and transcriptomes of the original hek293 along with five progeny cell lineages were analyzed ( fig. 1a ). the overall comparison of genomic and transcriptomic profiles confirmed the picture of clonally diverged progeny cells as compared to the parental hek293 ( fig. 1b, c ). as expected, there was a high degree of genomic and transcriptomic similarities of the freestyle 293 - f and 293 - f cell lines ( fig. 1b – d ).