BioSample

Both wildtype (target) and ngn3-/- (driver) cDNA libraries were amplified. The ssDNA from the wildtype cDNA library was prepared by infecting with M13KO7 and blocked with 5'GCGGCCGCT(15) oligo before hybridization. SalI digested dsDNA from the ngn3-/- cDNA library was used to synthesize biotinylated RNA with T7 RNA polymerase. Subtractive hybridization was performed at 42oC in 80% formamide, 100 mM HEPES pH 7.5, 2mM EDTA and 0.2% SDS. Streptavidin was used to remove clones common between the two libraries. After repair of the subtracted ssDNA with 5'GCGGCCGCT(15) oligo using Taq polymerase and Vent polymerase, the subtracted cDNA library was digested with SpeI and electroporated into DH12S cells (LTI).