BioSample

Total RNAs were extracted from 5 EPC. The double-stranded cDNA was synthesized by Gibco's kit with an Oligo(dT) primer [NotI primer-adapter from GibcoBRL] [5'-pGACTAGTTCTAGATCGCGAGCGGCCGCCCTTTTTTTTTTTTTTT-3'] from 0.8ug of mRNA . The double-stranded cDNAs were treated with T4 DNA polymerase and purified by ethanol-precipitation. The cDNAs were ligated to Lone-linker LL-Sal3 (include Sal1 sequence). The cDNAs were purified by phenol/chloroform and separated from free linkers by Centricon 100. Then, cDNAs were amplified by long-range high fidelity PCR using Takara's Ex Taq polymerase. Then, the cDNAs were purified by phenol/chloroform and by Centricon 100. The cDNAs were digested with SalI and NotI enzymes. Then, the cDNAs were size selected by Gibco's Size Fractionation Column. The cDNAs were cloned into SalI/NotI site of pSPORT1 plasmid vector. The DH10B E. coli host was transformed with the ligation mixture by chemical method. The library was constructed by Xiaohong Wang.