1. Mix
5 µg total RNA with oligo dT magnetic beads
2. Synthesize double-strand cDNA
3. Digest with NlaIII to form one end of the
tag
4. Divide in half and ligate 40 bp adapters (A
and B)
containing the recognition sequence for the
type-
II restriction enzyme BsmF 1
5. Cleave with BsmF 1 to form ~ 50 bp tag (40
bp
adaptor/13 bp tag)
6. Fill in 5' overhangs and ligate to form a ~
100 bp
ditag
7. PCR amplify using ditag primers 1 and 2
8. Cut 40 bp adapters with Nla III to release
the 26
bp ditag
9. Ligate ditags to form concatemers
10. Clone and sequence
Serial Analysis of Gene Expression (SAGE)