English: Schematic overview of cap analysis gene expression (CAGE). First, mRNAs are reverse transcribed in the presence of trehalose and sorbitol (allow reaction to occur at higher temperatures so secondary RNA structures can be relaxed and transcribed). Next, the 5’ caps of mRNA are biotinylated, RNase1 digests any ss-RNA, and full-length cDNA-RNA hybrids are captured using streptavidin beads (cap trapper method). cDNA is released from RNA, and a ds-CAGE linker is ligated to the ss-CDNA. The linker is biotinylated, and has cut sites for restriction enzymes. Synthesis of the second cDNA strand then occurs, after which, it is digested by a restriction enzyme, such as Mme1, leaving a 20-26 bp CAGE tag. A second linker is ligated onto CAGE tag. Next, the CAGE tags can be amplified, and sequenced.