BioSample

First strand cDNA synthesis was primed using oligo-dT on magnetic beads with an additional primer 5'-gcggccgctaatacgactcacta-taggg-3'. Second strand synthesis was a 3-cycle PCR using the primers 5'-ggccgctaatacgactcactatag-3' and 5' -aagcagtggtaacaacgcagagtacttt-ttttttttttttvn-3'. cDNA was subsequently amplified in a 7-cycle PCR with the following primers: 5'-ggccgctaatacgactcactatag-3' and 5'-aagcagtggt-aacaacgcag. Deoxy-UMP adaptors were added in a third PCR (5 cycles) and the primers 5'-caucaucaucauggccgctaatacgactcactataggg-3' and 5'-cuacuacuacuaaagcagtggtaacaacgcagagtac-3'. Ends were treated with uracil DNA glycosylase and product with 3' overhangs was annealed to complementary ends of pAMP1. Insert can be excised using EcoRI and NotI. Library constructed by Joe Barnes and Steve Johnson (Washington University).