Name: NIA Mouse Undifferentiated Embryonic Stem (ES) Cell cDNA Library (Long 1) Library ID: 2102 Organism: Mus musculus Strain: 129/Sv x 129S1/Sv-p<+> Tyr-c<+> Age: 0 Organ: embryonic stem cell Tissue: undifferentiated embryonic stem cells Host: DH10B Vector: pCMV-SPORT6 Vector type: phagemid Insert digest: 5' SalI/NotI 3' Stop Codon Status: without Description: Mouse cDNA project by the Laboratory of Genetics, National Institute on Aging (NIA), Intramural Research Program, NIH (http://lgsun.grc.nia.nih.gov/cDNA). This is a long-transcript enriched cDNA library (Ref. Genome Res. 11: 1553-1558 (2001). [PMID: 11544199]). Total RNAs were obtained from Dr. Kenneth R. Boheler (National Institute on Aging, USA). ES cells were cultured without feeder cells in the presence of LIF and BRL-conditioned media. Double-stranded cDNAs were synthesized with an Oligo(dT) primer [Invitrogen: 5'-pGACTAGTTCTAGATCGCGAGCGGCCGCCCTTTTTTTTTTTTTTT-3'] from 14.2 5g of total RNA, treated with T4 DNA polymerase, and purified by ethanol-precipitation. The cDNAs were ligated to Lone-linker LL-Sal4, purified by phenol/chloroform, and separated from free linkers by Centricon 100. Then, the cDNAs were amplified by long-range high fidelity PCR using Ex Taq polymerase (Takara) with a primer Sal4-S. The products were purified by phenol/chloroform and Centricon 100. The cDNAs were digested with SalI and NotI enzymes and cloned into SalI/NotI site of pCMV-SPORT6 plasmid vector. The DH10B E. coli host was transformed with the ligation mixture by the standard chemical method. The average insert size is about 2.4 kb. The library was constructed by Yulan Piao.