Figure 4

Flow-sheet of SAGE. mRNAs are captured on oligo(dT) magnetic beads (open ovals). Double stranded cDNAs are synthesized. They are digested with Nla III. The product is divided into two halves. These are ligated to 40 bp adapters AR and BR, respectively. Both adapters contain a sequence R that is a recognition site for the restriction enzyme Bsm FI. This cuts a 14 bp sequence 3' of the site, forming a 10 bp tag. After cleavage with Bsm FI, the tags are ligated to form a product containing a ditag (the points of ligation are denoted by filled circles). This is amplified using primers complementary to A and B. The AR and BR adapters are cut away with Nla III to release a ditag. These are ligated to form concatemers containing multiple ditags. The concatemers are cloned and sequenced.