To perform RNA sequencing, RNA is extracted from cells or tissues and reverse transcribed to cDNA (A). The cDNA is then digested into fragments, while the fragment length may vary depending on sequencing machine specifications (B). Each cDNA fragment is sequenced in a high-throughput manner to obtain short sequences termed “short sequenced reads” (SSRs). SSRs are aligned to a reference genome to produce a genome-scale transcription map (C). Transcript expression is calculated based on the number of reads aligned to each gene (D).