Next Generation Sequencing - An Analysis of the Role of Human-Specific Isoform transcription factor Oct-1 in Nerve Cell Differentiation
The aim of this study is to compare the NGS-derived profiling of human transcriptome (RNA-seq) in wild-type neuroblastoma cells with neuroblastoma transcript with a high level of expression of Oct-1 transcription factor and evaluate the effect of Oct-1 transcription factor on the regulation of nerve cell differentiation. Methods: Human mRNA profiles of 16-day differentiating wild-type neuroblastoma IMR32  and neuroblastoma IMR32 with overexpression primate-specific isoform of transcription factor Oct-1 were generated by deep sequencing, in triplicate, using Illumina NovaSeq. Mapping reads to the human genome (hg38) using hisat program. On average, about 89-90% of all received data was uniquely aligned in each library. The htseq-count utility calculated the number of reads that were mapped to known genes (ncbi - entrezID). The obtained values ??(cpm - countpermillion) for each gene for each library were combined into one matrix for further analysis. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the human genome (hg38). Filtration, normalization by the method (TMM), variance estimation and evaluation of differentially expressed genes were performed in the edgeR module. Genes in which cpm did not exceed 1 in any three libraries were considered low-expressing. After filtering the low-expressing genes, 15,108 entries remained. RNA-Seq data confirmed that approximately 8% of the transcripts showed differential expression between the WT and Oct-1 overexpressed differentiating neuroblastoma cells, with FDR<0.01 (p-value adjusted for multiple testing). Overall design: Human neuroblastoma mRNA profiles of 16-days differentiating wild-type neuroblastoma IMR32  and neuroblastoma IMR32 with overexpression primate-specific isoform of transcription factor Oct-1Z. Differentiation in MEM, 10% FCS, 2.5 mkM BrdU.