G-quadruplexes (G4) are special guanine-rich nucleotide structures that are made by Hoogsteen hydrogen bonds in DNA or RNA. They have become important factors in the complicated process of regulating gene expression. The action of G4 structures on genes is implicated in various diseases, including cancer and neurodegenerative disorders. Among the key regulators of G4 structures is DHX36, a versatile RNA and DNA helicase known for its ability to unwind G4 formations. In this study, we examined in detail how DHX36 influences gene expression through regulating the action of G4 using both wild-type and DHX36 knocked-out Jurkat cell lines. Using cutting-edge genomics and bioinformatics tools like edgeR, DEseq, and salmon, we performed a thorough investigation of the gene expression levels in these cell lines. Our findings demonstrated the subtle regulatory role of DHX36 in G4-mediated gene expression. The knockout cell line for DHX36 showed a clear upward trend in comparison to the wild-type cell line. Our findings demonstrate that DHX36 plays a crucial role in controlling the extent to which G4 structures affect gene expression. Moreover, the outcomes generated by different bioinformatics approaches can differ due to the computing matrices utilized in each approach.