Name: NIA Mouse Embryonic Germ Cell cDNA Library (Long, subtracted) Library ID: 2104 Organism: Mus musculus Strain: C57BL/6 Gender: male Age: 0 Stage: embryo Organ: germ cell 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]). EG cells were obtained from Dr. Brigid L.M. Hogan and RNA was prepared by Dr. Mark G. Carter (NIH/NIA-IRP). EG cells were cultured at 37? C, 5% CO2 in DMEM supplemented with 15% ES cell-qualified FBS, 0.1mM non-essential amino acids, 2 mM glutamine, penicillin/streptomycin, 1 mM sodium pyruvate, 0.1 mM beta-mercaptoethanol, and 10^7 units of LIF per liter. Double-stranded cDNAs were synthesized with an Oligo(dT) primer [Invitrogen: 5'-pGACTAGTTCTAGATCGCGAGCGGCCGCCCTTTTTTTTTTTTTTT-3'] from 2.5 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 double digested with Not1 and Sal1 enzymes, then purified by phenol/chloroform and Centricon 100. The cDNA mixture was subjected to a special subtraction procedure by Dr.Kazuhiro Kondo at AISIN Cosmos. Then the subtracted cDNAs were 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 2.2kb. The library was constructed by Yulan Piao and Kazuhiro Kondo.