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This normalized library was constructed from 1 million independent clones. cDNA synthesis was initiated using an oligo(dT) primer, using methylated C in the first strand synthesis reaction. Following this first strand reaction, double-stranded cDNA was blunted, ligated to NotI adapters, digested with EcoRI, size-selected, and cloned into the NotI and EcoRI compatible sites of a custom modified MCS of the pBluescript (KS+) vector. The library was normalized in 2 rounds using conditions adapted from Soares et al., PNAS (1994) 91: 9228-9232 and Bonaldo et al., Genome Research 6 (1996): 791, except that a significantly longer reannealing hybridization was used.
Nucleotide
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