Document Actions

Preparation of a SAGE Library

First proposed by Velculescu and colleagues in 1995, SAGE libraries are collections of thousands of small DNA "tags", each of which represents a distinct mRNA transcript. Sequencing the tags and identifying the genes from which they were derived allows the creation of a quantitative transcriptional profile of the tissue.

Instructions: Click on the name of the step to view the diagram for it.

1. Purify total RNA.
2. Reverse transcribe poly-A mRNA to cDNA. Bind to magnetic streptavidin beads.
3. Cut with anchoring enzyme (e.g. NlaIII).
4. Divide digests into two pools. Attach linkers 1 and 2.
5. Cut with type IIS tagging enzyme and fill in ends.
6. Ligate into ditags, amplify by PCR.
7. Cut off linkers with anchoring enzyme.
8. Concatemerise ditags.
9. Clone concatemers into sequencing vectors (e.g. pZero) and sequence.