What are the key steps involved in RNA-Seq?

The key steps involved in RNA-Seq are: RNA extraction, RNA fragmentation, cDNA generation, library preparation, and sequencing on a NGS platform to obtain continuous sequence data. During RNA extraction, RNA is isolated from the sample of interest. To ensure a successful RNA-seq experiment, the RNA should be of sufficient quality to generate a library for sequencing. Following RNA extraction, the isolated RNA is then fragmented into smaller pieces and reverse transcription is then carried to convert the fragmented RNA into cDNA. The next step is library preparation, which involves ligating specialized adapters to both fragment ends. These adapters possess the full complement of sequencing primer hybridization sites. The next step is sequencing, and the prepared cDNA libraries are loaded onto a high-throughput platform for NGS sequencing. NGS sequencing produces millions of short sequence reads, typically ranging from 50 to 300 base pairs in length. The generated sequence reads are then mapped to a reference genome for identification of transcripts. The abundance or expression level of each transcript can then be estimated by counting the number of aligned reads which map to specific regions of the reference genome.