Transcriptome, the functional element of the genome, is comprised of different kinds of RNA molecules such as mRNA, miRNA, ncRNA, rRNA, and tRNA to name a few. Each of these RNA molecules plays a vital role in the physiological response, and understanding the regulation of these molecules is extremely critical for the better understanding of the functional genome. RNA Sequencing (RNASeq) is one of the latest techniques applied to study genome-wide transcriptome characterization and profiling using high-throughput sequenced data. As compared to array-based methods, RNASeq provides in-depth and more precise information on transcriptome characterization and quantification. Based upon availability of reference genome, transcriptome assembly can be reference-guided or de novo. Once transcripts are assembled, downstream analysis such as expression profiling, gene ontology, and pathway enrichment analyses can give more insight into gene regulation. This chapter describes the significance of RNASeq study over array-based traditional methods, approach to analyze RNASeq data, available methods and tools, challenges associated with the data analysis, application areas, some of the recent advancement made in the area of transcriptome study and its application
