Rowayna ShouibGary EitzenRineke Steenbergen2025-05-132025-05-132025-05-05Shouib, R., Eitzen, G., & Steenbergen, R. (2025). A guide to basic RNA sequencing data processing and transcriptomic analysis. BIO-PROTOCOL, 15(1370). https://doi.org/10.21769/bioprotoc.5295https://doi.org/10.21769/bioprotoc.5295https://repository.msa.edu.eg/handle/123456789/6414SJR 2024 0.557 Q2 H-Index 22RNA sequencing (RNA-Seq) has transformed transcriptomic research, enabling researchers to perform largescale inspection of mRNA levels in living cells. With the growing applicability of this technique to many scientific investigations, the analysis of next-generation sequencing (NGS) data becomes an important yet challenging task, especially for researchers without a bioinformatics background. This protocol offers a beginner-friendly step-by-step guide to analyze NGS data (starting from raw .fastq files), providing the required codes with an explanation of the different steps and software used. We outline a computational workflow that includes quality control, trimming of reads, read alignment to the genome, and gene quantification, ultimately enabling researchers to identify differentially expressed genes and gain insights on mRNA levels. Multiple approaches to visualize this data using statistical and graphical tools in R are also described, allowing the generation of heatmaps and volcano plots to represent genes and gene sets of interest.en-USBioinformaticsmRNANext-generation sequencing (NGS)RNA-SeqTranscriptomic analysisArticlehttps://doi.org/10.21769/bioprotoc.5295