title: Removal of polyA tails from full-length cDNA libraries for high-efficiency sequencing.
abstract: We have developed a method to overcome sequencing problems caused by the presence of homopolymer stretches, such as polyA/T, in cDNA libraries. PolyA tails are shortened by cleaving before cDNA cloning with type IIS restriction enzymes, such as GsuI, placed next to the oligo-dT used to prime the polyA tails of mRNAs. We constructed four rice Cap-Trapper-selected, full-length 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 for direct sequencing and transcriptional sequencing without the slippage observed for libraries prepared with currently available methods, thus improving sequencing accuracy, operations, and throughput.
authors.affiliation: Genome Exploration Research Group, RIKEN Genomic Sciences Center, Kanagawa, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Shibata
authors.forename: P
authors.initials: P
authors.lastname: Carninci
authors.forename: K
authors.initials: K
authors.lastname: Sato
authors.forename: N
authors.initials: N
authors.lastname: Hayatsu
authors.forename: T
authors.initials: T
authors.lastname: Shiraki
authors.forename: Y
authors.initials: Y
authors.lastname: Ishii
authors.forename: T
authors.initials: T
authors.lastname: Arakawa
authors.forename: A
authors.initials: A
authors.lastname: Hara
authors.forename: N
authors.initials: N
authors.lastname: Ohsato
authors.forename: M
authors.initials: M
authors.lastname: Izawa
authors.forename: K
authors.initials: K
authors.lastname: Aizawa
authors.forename: M
authors.initials: M
authors.lastname: Itoh
authors.forename: K
authors.initials: K
authors.lastname: Shibata
authors.forename: A
authors.initials: A
authors.lastname: Shinagawa
authors.forename: J
authors.initials: J
authors.lastname: Kawai
authors.forename: Y
authors.initials: Y
authors.lastname: Ota
authors.forename: S
authors.initials: S
authors.lastname: Kikuchi
authors.forename: N
authors.initials: N
authors.lastname: Kishimoto
authors.forename: M
authors.initials: M
authors.lastname: Muramatsu
authors.forename: Y
authors.initials: Y
authors.lastname: Hayashizaki
mesh_terms: D000818:Animals; D018076:DNA, Complementary / Q000737:chemistry*; D015723:Gene Library; D051379:Mice; D008810:Mice, Inbred C57BL; D011061:Poly A / Q000737:chemistry*; D017422:Sequence Analysis, DNA / Q000379:methods*; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D011061:Poly A