BioSample

The library was constructed by reverse transcription of 1 ug mRNA using the oligo dT primer GCGGCCGCCC(T)20 and an RNaseH + MMLV reverse transcriptase. Second strand synthesis was carried out by standard methods. The cDNA was size selected by agarose gel for > 1.2 kb, digested with Not I and directionally cloned into the vector Express-1 at the SmaI/NotI sites. DNA from the primary library was used for in vitro transcription from the T7 promoter to produce biotinylated RNA transcripts. These biotinylated transcripts, along with blocking oligos to the poly-A, multiple cloning site and primer regions, were hybridized with single stranded circles produced by phagmeid production from the primary library to a Cot value of 10-20. Strepavidin/phenol extraction was utilized to remove DNA:RNA hybrids leaving un-hybridized single stranded circles which were repaired by primer extension and transformed back into E. coli resulting in the normalized library. Average insert size 2.0 kb. 3' linker/adaptor sequence GCGGCCGCCC(T)20. This libary was constructed by Agencourt Bioscience.