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Total RNA was isolated from pooled brain tissues using modified Trizol method (Invitrogen), then mRNA was extracted and purified from total RNA using Oligotex mRNA kit (Qiagen). cDNA was prepared from 5 micrograms of mRNA and directionally ligated into the pBluescript II SK (Sfi I) vector using SMARTTM cDNA library construction kit according to manufacturer's instructions (Clontech). Plasmid DNA was then transformed by electroporation into DH10B cells (Invitrogen) for propagation. Normalization was applied according to published methods [Bonaldo M.F. et al. (1996) Genome Research 6(9):791] in order to reduce the abundance of highly expressed transcripts.
Nucleotide
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