We have developed a method to overcome sequencing problems caused by the pr
esence of homopolymer stretches, such as polyA/T in cDNA libraries. PolvA t
ails are shortened by cleaving before cDNA cloning with type IIS restrictio
n enzymes, such as GsuI, placed next to the oligo-dT used to prime the poly
A tails of mRNAs. We constructed four rice Cap-Trapper-selected. full-lengt
h normalized cDNA libraries, of which the average residual polyA tail was 4
bases or shorter in most of the clones analyzed. Because of the removal of
homopolymeric stretches, libraries prepared with this method can be used f
or direct sequencing and transcriptional sequencing without the slippage ob
served for libraries prepared with currently available methods, thus improv
ing sequencing accuracy, operations, and throughput.