1. Create ds cDNA from expressed mRNA.
2. Chop up cDNA with DpnII (GATC recognition sequence), then select for the 3’ most fragment that extends to the poly (A) addition site.
3. Tag each cDNA at its poly (A) end with a 32-base nucleotide tag consisting of the bases A, T, G and comprising eight different four-letter nucleotide words, for a total of 16.7 million different tags. To be 99 percent confident that each cDNA will be tagged with a unique tag, only 1 percent of the tags are used at once.
4. Amplify the tagged cDNA fragments using PCR.
5. Mix in the tagged fragments with Lynx’s microbeads, each of which has 32-base antitags radiating out from it. The antitags, made out of the 16.7 million different 32-base pair permutations of C,A, and T, are designed to be complementary to the tags. Each bead has at least 100,000 identical antitags on it, which are synthesized on the bead base by base.
6. The tags hybridize to the antitags. All of the tags and antitags have the same melting temperature, but the nearest neighbor mismatch for each tag has a Tm several degrees different than the perfect match. So temperatrue control limits non-specific binding.
7. The DNA-laden beads are rolled into a flow cell to create a monolayer, and then into a sequencing instrument. The sequencing reagents are added to the surface of the beads, and flow through the flow cell while the beads stay still.
8. The DNA on the beads is sequenced four bases at a time for 17 bases toward the center, the minimum number of bases to determine a unique sequence.
9. These sequences are identified and counted. The level of expression for one gene is expressed as no. identical signatures/total no. transcripts analyzed.