Figure 2.

Outline of the first subtractive step. Synthetic RNAs were produced by run-off in vitro transcription from a 3′-end-specific cDNA library (HM3). The resulting RNA molecules are nearly uniform in length (400–600 nt) and their composition reflects the relative abundance of each cDNA in the starting library. Super-prevalent RNAs are shown as black wavy lines. We designed DNA oligonucleotides complementary to the 3′-UTR of the super-prevalent mRNAs, then RNAs and DNA oligonucleotides were incubated in the presence of RNase H, which specifically degrades the RNA moiety in a DNA:RNA hybrid. The undigested RNAs were purified from preparative gels, reverse transcribed and cloned in plasmid vectors.