EFFECTS OF DUPLEX-SPECIFIC NUCLEASE ON HUMAN CELL GENE EXPRESSION PROFILING USING RNA-SEQ

RNA-seq is a next-generation sequencing technology able to characterize thoroughly gene expression profiles and to perform very accurate differential gene expression (DE) analysis. In some experimental conditions RNA-seq displays limitations, such as in studies with few, but extremely high, transcript expression. High expression of few genes could both influence the measurement of relative expression of less expressed genes, as well as reduce the estimate accuracy of gene expression levels of low expressed genes. Therefore, it would be convenient to apply a method by which variability of the expression level of each transcript does not depend on few highly expressed transcripts. Thus a cDNA depletion method for the most represented transcripts called duplex-specific nuclease (DSN) has been effectively used in this study. DSN technique is based on cDNA denaturation, slow hybridization and degradation of double-stranded DNA. To investigate the performances of DSN treatment on different cell types we studied three different cell types: whole blood, monocytes and keratinocytes. From each cell type two cDNA libraries were produced (untreated libraries), for a total of 6 libraries. A half of each library was then treated with DSN, producing a total of 6 DSN treated libraries. Overall, 12 cDNA libraries (6 untreated and 6 treated) were prepared. Afterward, the prepared libraries underwent RNA-seq. Quality control checks were carried out and the data analysis was completed. In blood tissue the most abundant transcripts in both DSN-treated and DSN-untreated samples corresponded to globins (HBA2, HBA1 and HBB), accounting for ~70% of total reads (i.e. short sequences of nucleotides). In this sample maximum effects of DSN treatment were observed. Transcripts found to be the most expressed in monocyte sample were ~10 times smaller than globins expression in blood sample. Gene expression of keratinocyte libraries was not influenced by DSN treatment. DSN treatment has been able to strongly reduce globins expression. DSN treatment also reduced, even if with a smaller effect, the most expressed genes in monocyte sample. We conclude that DSN treatment is suitable for samples presenting highly-expressed genes as it allows to observe more DE genes, compared to the untreated RNA-seq samples.