Name: NIA Human H1 Embryonic Stem Cells cDNA Library (Long) Library ID: 2045 Organism: Homo sapiens Age: 0 Stage: embryo Organ: embryonic stem cell Tissue: undifferentiated ES cell line H1 Host: DH10B TonA Vector: pCMV-SPORT6 Vector type: phagemid Insert digest: 5' SalI/NotI 3' Stop Codon Status: without Description: This is a long-transcript enriched cDNA library (Ref. Genome Res. 11: 1553-1558 (2001). [PMID: 11544199]). Undifferentiated human ES cell line H1 was obtained from WiCell Research Institute, Inc., Madison, Wi, cultured according to their instructions, and prepared for RNA extraction by Drs. Drs. Irene Ginis and Mahendra S. Rao. Briefly, cells were cultured on mouse embryonic fibroblast (MEF) feeders in ES cell medium consisting of DMEM/F12 (Invitrogen/GIBCO 11330-032) supplemented with 20% Knockout Serum Replacement, 100 mM MEM nonessential amino acids, 0.55 mM beta-mercaptoethanol, 2 mM L-glutamine, antibiotics/antimycotics, and with 4 ng/ml human basic fibroblast growth factor (bFGF) (all from Invitrogen/GIBCO). MEF derived from E12.5 mouse embryo were purchased from StemCell Technologiea, Inc. Vancouver, Canada. MEF were expanded on 0.5% bovine gelatin-coated dishes in DMEM medium (cat#11965-092) supplemented with 10% FBS (heat inactivated, Hyclone cat#3007103), 2 mM glutamine and 100 mM MEM non-essential amino acids. Subconfluent MEF cultures were treated with 1 mg/ml Mitomycin C (Sigma) for 3 hours to arrest cell division, trypsinized and plated at 0.2x105/cm2 onto 5% bovine collagen-coated dishes overnight. Feeders were washed twice with PBS; and then incubated in ES cell medium for at least 1 hour before plating ES cells. MEF''s of passage 2-3 were used as feeders. ES cells plated on top of MEF feeders were cultured at 5% CO2/5% O2 humidified tissue culture incubator from Thermo Forma. They formed round colonies with defined edges and were positive for alkaline phosphatase and SSEA-4. When confluent (18-10 days after plating) ES cells were treated with 1 mg/ml collagenase, type IV (Invitrogen/GIBCO) for 5-10 min and gently scraped off with 5 ml pipette. Cells from 4X6cm dishes were spun at 500 rpm for 3 min and the pellet was used for RNA purification. RNA was purified with TRIzol Reagent from Invitrogen. Double-stranded cDNAs were synthesized with an Oligo(dT) primer [Invitrogen: 5''-pGACTAGTTCTAGATCGCGAGCGGCCGCCCTTTTTTTTTTTTTTT-3''] from 3.45g 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 extraction, and separated from free linkers by Centricon-100 column. 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 extraction and Centricon-100 column. 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 3.6kb. The library was constructed by Yulan Piao. Note: this is a NIH_MGC Library.